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Eimeria acervulina, E. brunetti, E. maxima, and E. necatrix: Low doses of oocysts to immunize young chickens
40,250-260
EXPERIMENTALPARASITOLOGY
(1976)
Eimeria acervulina, E. brunetti, E. maxima, and E. necatrix: Low Doses of Oocysts to Immunize Young Chickens HELEN Central Veterinary Fisheries and
(Accepted
E. HEIN
Laboratory, Ministry of Agriculture, Food, Weybridge, Surrey, England
for publication
24 November 1975)
HEIN, H. E. 1976. Eimeria aceroulina, E. brunetti, E. maxima, and E. necatrix: Low doses of oocysts to immunize young chickens. Experimental Parasitology 40, 250-260. Resistance to reinfection varied with the species of Eimeria and with the number of oocysts in the inoculum. Chickens immunized with doses of 20,000 and 80,000 oocysts of E. acewulina, 312 and 1250 oocysts of E. brunetti or E. necatrix, or 1250 and 5000 oocysts of E. maxima at 2 and 4 weeks of age, respectively, were almost completely immune to a challenge dose at 6 weeks of age. Resistance was slightly less in chickens immunized with 1250 and 5000 oocysts of E. aceruulina or 312 and 1250 oocysts of E. maxima. Birds given three immunizing infections of 1250, 5000, and 20,000 oocysts of E. maxima were completeIy immune 8 weeks after the last dose. Resistance was slightly less in birds immunized with similar doses of E. brunetti or E. necatriz. Doses of 20,000, 80,000, and 320,000 oocysts appeared necessary to confer a high level of immunity to E. acervulina. More than three low doses of oocysts appear necessary to induce a complete and enduring immunity against a high challenge for E. acervulina, E. brunetti, and E. necatrix. Higher immunizing doses would not be satisfactory due to the pathogenic effects of the coccidia after the initial infection. INDEX DESCRIPTORS: Eimeria aceroulina; Eimeria brunetti; Eimeria maxima; Eimeria necatrix; Oocysts; Immunity; Chickens.
INTRODUCTION
Dickinson ( 1941), Rose and Long (1962), and Hein ( 1968b) have shown that when repeated coccidial infections are administered to young chickens, a high level of immunity develops. Dickinson (1941) inoculated chickens with E. acervulinu and gave daily doses of oocysts over a period of 5 to 10 days. He showed that there was no change in body weight when birds were heavily challenged 3 to 9 weeks later. Rose and Long (1962) inoculated replicate groups of chickens with two or three rising doses of oocysts of E. acervulina, E. maxima, E. tenella, or E. necatrix, with an interval of 7 days between each infeotion. 250 Copyright
@ 1976
by Academic
Press,
Inc.
All rightr of reproduction in any form reserved.
Their birds were completely protected when challenge’d 38 and 84 days after the initial immunizing infection. Hein (196&b) administered lower doses of E. acervulinu with an interval of 14 days between each infection to replicate groups of 2-week-old chickens and showed that a high level of resistance was conferred against challenge 28 days after the initial infection. Little data are available however on the immunogenic potential of low doses of oocysts, the duration of immunity, or differences in the immunogenicity of different species. Dickinson (1941) and Rose and Long (1962) tended to use high doses of oocysts for immunization with only a short interval of time between each infection and
EIMEFUA
OOCYSTS
TO
IMMUNIZE
their results may have been modified by interference between succeeding infections. Observations on the immunogenic potential of E. maxima appear to be in conflict because the results of Rose and Long ( 1962) do not support the conclusion of Long (1962) that immunity to E. maxima was of short duration. Furthermore, Hein ( 1968b) found that immunity appeared lower in chickens which had received the higher immunizing infections with E. acervulina which was contrary to expectation and requires confirmation. The earlier experiments were carried out under a multiplicity of experimental conditions so that the results are not directly comparable. The present study was undertaken therefore to obtain data on the immunogenic potential of E. acervulina, E. brunetti, E. maxima, and E. necatrix under precisely defined conditions which would provide standards enabling comparisons between species and strains to be made. MATERIALS
Experimental
AND
METHODS
Birds
White Link hybrid cockerel chickens (Sterling Poultry Products Ltd) were used. They were reared in isolation and transferred to the experimental unit 2 days before infection. They were housed under conditions to prevent reinfection and were fed ad libitum on a standard ration (Joyner and Davies 1960). The chickens were e-weeks-old when infected initially (Day 0). Parasitology The oocysts used in the experiments were from the same source as those used previousIy and were prepared under the same standard conditions (Hein 1968a, 1971, 1974). Fresh suspensions of oocysts were prepared for each infection. The age of the oocysts, calculated from the day of recovery from the feces, was 10 or 11 days on each occasion. Dilutions were made to
YOUNG
CHICKENS
251
give the appropriate number of sporulated oocysts ,in a volume of 1 ml on Day 0 for the 2-week-old chickens and in 2 ml on Days 14, 28 and 84 when the chickens were 4-, 6-, ‘and lCweeks-old. After infeotion, the average total daily oocyst output per bird was estimated in a similar manner to that described previously ( Hein 1968a). Experimental
Design
The chickens received either two immunizing doses of oocysts and a challenge infection on Day 28 or three immunizing doses of oocysts and a challenge infection on Day 84. An interval of 14 days was judged to be sufficient to avoid interference between successive infections. The number of oocysts administered in the immunizing and challenge infections are shown in Table I. Low doses of oocysts were chosen for immunization to minimize the detrimental effect on the host and to allow maximum development of the parasite without overcrowding which might modify the immunogenic potential of the coccidia. High doses of oocysts were selected for challenge to produce typical signs of coccidiosis in susceptible chickens. Two days before the initial infection the chickens were weighed and randomized into groups of 10 chickens with approximately equal mean weights. The chickens were weighed daily after each immunizing and challenge infection and total daily oocyst counts were ‘made throughout the course of the experiment. Noninfected chickens served as a control group throughout the experiment. The pathogenicity of the challenge inocula was confirmed in replicate groups of chickens on each occasion. A series of analyses of variance on the daily weights and weight gains of individual birds was carried out, and mean values were compared to assess the statistical significance of the differences between groups.
252
HELEN E. HEIN TABLE: I The Doses of Eimeria spp Oocysts Administered to Young Chickens in the Immunizing and Challenge Infections
Species
E. acervulina
E. brunetti
E. maxima
E.
necutrix
Immunizing
infections
Challenge infections
Day 0
Day 14
Day 28
Day 28
1,250 20,000 320,000 1,250 20,000 320,000
5,000 80,000 1,280,000 5,000 80,000 1,280,000
-
5,120,OOO
312 1,250
1,230 5,000
-
312 1,250
1,250 5,000
5,000 20,000
312 1,250
1,250 5,000
-
1,250 20,000
5,000 80,000
20,000 320,000
1,250 312
5,000 1,250
- 5,000
1,250
5,000
20,000
20,000 320,000 6,120,000
Day 84
10,240,OOO
320,000 i 1,28O,OC^ I” 320,000 1,280,000 320,000 >
320,000
equalled that of the noninfected controls and by Day 26 there was no significant The effects of the immunizing infections difference in mean weight between these are shown in Table II and Figs. la, lb, lc, and Id, and those of the challenge infec- and the birds infected with the two lower tions in Tables III and IV and Figs. 2a, 2b, doses of oocysts (Fig. la). However, the 2c, 2d and 3a, 3b, 3c, and 3d. mean weight of the birds inoculated with the highest doses of oocysts was signifiE. acervulina cantly lower (P
EIMERL4
OOCYSTS
TO
IMMUNIZE
infection was the presence of oocysts in the feces during the patent phase of the disease. In birds immunized with the lowest doses of oocysts, total output was approximately 2 and 77 million oocysts per bird, respectively, whilst in the chickens immunized with either of the higher doses of oocysts the number was too low to count (Tables III and IV). E. brunetti After the first infection, morbidity and retardation of growth were marked on Days 5 and 6, ,the effect being most severe in the group receiving the higher dose of 1250 oocysts in which one bird died on
YOUNG
Day 6 (Table II, Fig. lb). The mean weight gains were significantly lower in the infected chickens than in the noninfected controls ( P
TABLE The Infection
Pathogenic
Effects
1
2
II
of the Immunizing 3
1
253
CHICKENS
Infections 2
with 3
Eimeria
spp
1
2
3
320,000 +” -
1,280,OOO -
5,120,OOO -
+
+
E. acervulina Dose Clinical signsa Mortality Mean y0 wt lossa Oocysts~
1,250 4 19
5,000 -
+”
20,000 -
20,000 -
+
320,000 -
1,250 ++ l/10 22 20
5,000 -
20,000 -
1,250 -
5,000 -
20,000 -
8 73
80,000 -
19 30
E. brunetti Dose Clinical signs Mortality Mean y0 wt loss oocysts
312 +i-
5,000 -
14 20
E. maxima Dose Clinical signs Mortality Mean $& wt loss Oocysts
312 10 3
1,250 -
-
11 7
20,000 + 18 5
80,000 -
320,000 -
E. necatrix Dose Clinical signs Mortality Mean y0 wt loss Oocysts
312 -
1,250
-
10
a Clinical signs : + denotes slight b Mean percentage loss in weight 0 Average total oocyst production too small to count.
5,000 -
1,250 12
5,000 -
20,000 -
anorexia, + + marked anorexia during immunization compared per bird in millions, + denotes
and depression. with noninfected control birds. oocysts found on salt flotation,
number
254
HELEN
A
E.ACERVUlINA
E. HEIN
E .BRUNETTI
lb
E MAXIMA
DAYS AFTER INOCULATION FIG. 1. The mean weight in grams of chickens after inoculation with the first and second immunizing doses of Eimeriaspp. oocysts. Key: -ononinfected chickens; -n312 + 1250 oocysts; -x-1250 + 5000 oocysts; -A-20,000 + 80,000 oocysts; -W-320,000 + 1,280,OOO oocysts.
2 days before challenge on Day 82. Following challenge on Day 28 or Day 84, there was no indication of morbidity or growth retardation in the immunized chickens (Tables III and IV, Figs. 2b and 3b). A few oocysts were detected in the feces of the immunized birds but the number was too low to count (Tables III and IV). E. maxima After the first infection, slight retardation of growth was noted on Day 7 in the chick-
ens inoculated with 312 oocysts (P
EIMERIA
OOCYSTS
TO
IMMUNIZE
with 1250 or 20,000 oocysts was slightly lower than that of the noninfected birds (P
Pathogenic
Effects
of Eimeria
255
CHICKENS
mean weight between the immunized birds and the noninfected chickens were not significant. E. necatrix After the first infection, there was no sign of morbidity or retardation of growth in either group of chickens. The average total production of oocysts was 10 and 12 million oocysts per bird, respectively (Table II). After the second and third infections, no oocysts were detected in the feces. Following challenge on Day 28, there was no evidence of infection in either group of immunized chickens (Table III, Fig. 2d). After challenge on Day 84, no sign of morbidity or growth retardation was noted in the immunized chickens, a few oocysts were detected in the feces of each group but the number was too small to count (Table IV, Fig. 3d). The pathogenicity of the challenge infections on Day 28 and Day 84 was con-
TABLE The
YOUNG
III
spp. Challenge
Infections
Administered
on Day
E. acervulina Dose Immunizing dose Clinical signs” Mortality Mean To weight oocysts x 106
None ++” lossb
8 75
5,120,OOO
1,250 5,000 -
E. brunetti oocysts
20,000 80,000 -
-
+”
2
Dose 320,000 1,280,OOO +
None +++” 30 10
E. maxiwra Dose Immunizing dose Clinical signs Mortality Mean y0 weight) Oocysts x 106
None +++ loss
21 6
320,000
28
320,000
oocysts
312 1,250 -
1,250 5,000 -
+
+
E. necatrix oocysts
312 1,250 -
1,250 5,000 -
+
-
Dose None ++++” lO/lO
@Clinical signs : + + marked anorexia and depression : + + + marked anorexia, stained feces : + + + + very high morbidity. 6 Mean ‘% weight loss after challenge compared with noninfected control chickens. c + denotes a few oocysts found on salt flotation, number too small to count.
320,000
oocysts
312 1,250 depression
1,250 5,000 and blood
256
HELEN
E. HFJN
TABLE
IV
The Pathogenic Effects of Eimeria spp. Challenge Infections Administered E.
acervulina
E. brunetti
Dose 10,240,OOO oocysts Immunizing doses
None
1,250 5,000 20,000
Clinical signs Mortality Mean y0 weight lossa oocysts x 106
+++” l/8
320,000
7
-
Dose 1,280,OOO oocysts 320,000
77
$6
None
5,120,000
-
T
5,000
+++ 4/8 26 44
E. maxima
Dose 1,280,OOO oocysts Immunizing doses
None
Clinical signs Mortality Mean ‘% weight loss oocysts x 106
+++ 16
1,250 5,000 20,000
20
20,000
E.
None
320,000
-
-
-
1,250 5,000 20,000
-
-
-
-
+
+
-
necatrix
Dose 320,000 oocysts
80,000
-
312 1,250
1,280,000
-
-
192
20,000
80,000
on Day 84
++++” lO/lO
312 1,250
1,250 5,000
5,000
20,000
-
-
+
+
a Mean y0 weight loss after challenge compared with noninfect,ed control chickens. b + denotes a few oocysts found on salt flotation, number too small to count. c Clinical signs : + + $ High morbidity : + + + 4 Very high morbidity.
feces of the immunized chickens after challenge on Day 84. Chickens inoculated with E. brunetti passed a few oocysts after each challenge infection suggesting that resistance was slightly lower in these birds. DISCUSSION With E. acervulina, approximately 2 milA very high level of resistance to reinfeclion oocysts per bird were produced after tion was conferred by multiple infections challenge on Day 28 which suggested that with relatively low doses of oocysts of E. the immunogenic potential of this species acervulina, E. brunetti, E. maxima, or E. was lower. This was confirmed by the relanecatrix. tively high output of 77 million oocysts Small differences were apparent in the after challenge on Day 84. immunogenic potential of these species of With all four species, even the lowest coccidia. In chickens immunized with stan- doses of oocysts produced an easily demdard doses of 1250 and 5000 oocysts and onstrated immunity. There was no clinical challenged on Day 28, or given a third dose response to any of the challenge infections. of 20,000 oocysts and challenged on Day However, in chickens immunized with the 84, no evidence of infection was detected smallest doses of E. maxima a few oocysts in the birds inoculated with E. maxima were detected in their feces after chalwhich indicated that these birds were com- lenge on Day 28, while in birds inoculated pletely immune. Resistance was also very with E. acervulina, oocyst production was high in birds receiving E. necatrix, al- demonstrably greater when they had rethough a few oocysts were present in the ceived the lowest immunizing doses, The firmed by the typical signs of acute coccidiosis with h6gh mortality in each group of susceptible chickens (Tables III and IV, Figs. 2 and 3).
ElIMERIA
OOCYSTS
TO
IMMUNIZE
results for these two species suggest that the level of immunity is influenced by the number of oocysts administered. These observations are in general agreement with ‘those of Rose and Long (1962) who reported that the immunizing activity of coccidia varied with the species of Eimeria, and with the work of Rose (1971) which indicated that in the case of E. maxima protection was dose dependent. Differences in the effects of the immunizing and challenge infections showed that
YOUNG
CHICKENS
257
the optimum number of oocysts for immunization varied with the species of Eimeria. When chickens received two immunizing infections followed by a challenge infection on Day 28, the reduced oocyst production after challenge in birds infected with 20,000 and 80,000 oocysts of E. acervulina indicated that these doses of oocysts will not give total immunity but will give protection against the clinical effects of a high challenge infection. The high mortality and marked weight loss associated *co
I
I
E BRUNETTI
2b
DAYS AFTER CHALLENGE FIG. 2. The mean weight in grams of immunized and susceptible chickens after Eimetia spp. challenge on Day 28. Key: -@-noninfected chickens; -Asusceptible chickens; -nimmunized 312 + 1250 oocysts; -Ximmunized 1250 + 5000 oocysts; -Aimmunized 20,000 + 80,000 oocysts; ---320,000 + 1,250,OOO oocysts.
258
HELEN
E.
HEIN xmo
?mw
E.BRUNETTI
E.ACERVULINA
2000
E .MAXIMA
E. NECATRIX
DAYS AFTER CHALLENGE FIG. 3. The mean weight in spp. challenge on Day 84. Key: immunized 312 + 1250 + 5000 immunized 20,000 + 80,000 + 5,120,OOO oocysts.
grams of immunized and susceptible chickens after Eimeria -o-- noninfected chickens; -Asusceptible chickens; -ooocysts; -Ximmunized 1250 + 5000 + 20,000 oocysts; --A320,000 oocysts; -H-immunized 320,000 + 1,280,OOO +
with infections of E. brunetti (Hein 1974) suggests that a dose of 312 followed by 1250 oocysts approaches the maximum tolerated by young chickens. The presence of a few oocysts after challenge in birds inoculated with similar numbers of oocysts of E. maxima indicated that the higher doses of 1250 and 5000 oocysts are necessary to obtain complete immunity. Both regimes used for E. necatrix gave good immunity against both challenge infections without obvious clinical effects in the birds. However, in view of the high pathogenicity
of this species of coccidia (Hein 1971) and the difference in susceptibility of chickens to infection associated with breed (Long 1968) it would seem wise to select the lower doses of 312 and 1250 oocysts for immunization. The results from groups given three immunizing infections and challenged on Day 84 were complementary to those in which birds received 2 immunizing doses of oocysts and a challenge on Day 28. However the experiments were not strictly comparable, so that further studies are necessary to establish the mini-
EIMEFUA
OOCYSTS
TO IMMUNIZE
mum number of doses of oocysts required to confer a high and enduring immunity. The virtual absence of oocysts in the droppings after the second immunizing infection suggests that a high level of immunity may arise from the initial infection and recent studies by Rose (1971) have indicated that with E. maxima this protection may be at its greatest on the 14th day after infection. This coincides with the time of the second immunizing dose of oocysts in the present study. In the experiments recorded by Rose and Long ( I962), high numbers of oocysts were produced in the feces of the birds after the second immunizing infection, and comparisons of the immunogenic potential of each species were based upon the observed oocysts production. The differences between their observations and those in the present experiments in which very little oocyst production was seen after the second infection, may have been due to the particular doses used and to the shorter time interval of 7 days which Rose and Long allowed between successive infections. Pout (1969) studied the changes in the intestinal mucosa following infection with Eimeria sp. and suggested that at least 14 days must elapse before the tissue regains its normal architecture; infections given within this intervaI may be modified by tissue changes. The level of immunity conferred by doses of 20,000 and 80,000 oocysts of E. acervuha appeared similar to that in birds inoculated with 80,000 and 160,000 oocysts of the same species in earlier studies (Hein 196813). Immunity was almost complete in birds infected with the highest doses of 320,000 and 1,280,OOO oocysts throwing considerable doubt on the previous results in which resistance appeared to decrease when the immunizing infections were increased from 80,000 and 160,000 to 320,000 and 640,000 oocysts (Hein 196813). Resistance in birds immunized with doses of 20,000, 80,000, and 320,000 oocysts of
YOUNG
CHICKENS
259
appeared comparable to that in birds given repeated daily infections with approximately the same total number of oocysts by Dickinson ( 1941). The presence of a few oocysts in the feces of 1the immunized birds after challenge indicated that immunity was lower than that obtained by Rose and Long (1962) with far higher doses of 500,000, 5,000,000, and 10,000,000 oocysts, when no evidence of infection was detected after challenge with 20,000,000 oocysts on both Day 38 and Day 84. Changes in body weight were not mentioned by these workers but dose response studies (Hein 1968a) suggest that the detrimental effects of the parasite would have been severe after the initial immunizing infection. This receives support from the marked growth retardation in birds inoculated with 320,000 oocysts in the current study. Thus it would seem preferable to employ low doses of oocysts for immunization ‘and to increase the number of infections to induce a high level of immunity and avoid growth retardation. The presence of oocysts in the feces of birds after challenge on Day 84 in birds immunized with oocysts of E. brunetti or E. necatrix suggests that with these species, more than three infections are required to induce a lasting complete immunity against a high challenge infection. Rose and Long ( 1962) immunized birds with doses of 500, 5000, and 50,000 oocysts of E. necatrix and found no evidence of infection when the same group of birds was challenged with 100,000 oocysts both on Day 38 and Day 84. Their birds had received, in effect, four immunizing infections at the time of the final challenge on Day 84. In the present experiment, immunizing infections were far lower, the challenge infection was higher and the time interval between immunization and challenge was greater. The results suggest that the large doses of E. necatrix used by Rose and Long (1962) are not necessary to produce an effective immunity to this species. E. acetwlina
260
HELEN
The high level of immunity in birds inoculated with E. maxima confirmed the observations of Rose and Long (1962) that this species has a high immunogenic potential. The results however throw considerable doubt on the observation of Long (1962) that immunity to E. maxima is of short duration. ACKNOWLEDGMENTS The author wishes to thank Dr. L. P. Joyner for help in the preparation of the paper and Miss N. Hebert for her statistical analysis of the results. She also thanks Mr. R. Sayer and Mr. P. Hall for their help in the preparation of Figs. 1-3 and Mr. C. Norton and Mr. P. Hall for their excellent technical assistance. REFERENCES DICKINSON, E. M. 1941. The effects of variable dosages of sporulated Eimeria acertiulina oocysts on chickens. Poultry Science 20, 413-424. Hum, H. E. 1968a. The pathogenic effects of Eimeria acervulina in young chicks. Experimental Parasitology 22, l-11. HEIN, H. E. 196813. Resistance in young chicks to
E. HEIN reinfection by immunization with two doses of oocysts of Eimeria acewulina. Experimental Parasitology 22, 12-18. HEIN, H. E. 1971. Pathogenic effects of Eimeria necatrix in young chickens. Experimental Parasitology 30, 321330. HEIN, H. E. 1974. The pathogenic effects of Eimeria brunetti in young chickens. Experimental Parasitology 36, 333-341. JOYNER, L. P., AND DAVIES, S. F. M. 1960. Detection and assessment of sublethal infection of Eimeria tenella and Eimeria necatrix. Experimental Parasitology 9, 243-249. LONG, P. L. 1962. Observations on the duration of acquired immunity of chickens to Eimeria maxima Tyzzer, 1929. Parasitology 52, 89-93. LONG, I’. L. 1968. The effect of breed of chickens on resistance to Eimeria infections. British Podtry Science 9, 71-78. POUT, D. D. 1969. Observations on coccidiosis of grazing lambs with special reference to the mucosal reaction of the small intestine. Ph.D. thesis, University of London. ROSE, M. E., AND LONG, P. L. 1962. Immunity to four species of Eimeria in fowls. Immunology 5, 79-92. ROSE, M. E. 1971. Immunity to coccidiosis: protective effect of transferred serum in Eimeria maxima infections. Parasitokgy 62, 11-25.
EXPERIMENTALPARASITOLOGY
(1976)
Eimeria acervulina, E. brunetti, E. maxima, and E. necatrix: Low Doses of Oocysts to Immunize Young Chickens HELEN Central Veterinary Fisheries and
(Accepted
E. HEIN
Laboratory, Ministry of Agriculture, Food, Weybridge, Surrey, England
for publication
24 November 1975)
HEIN, H. E. 1976. Eimeria aceroulina, E. brunetti, E. maxima, and E. necatrix: Low doses of oocysts to immunize young chickens. Experimental Parasitology 40, 250-260. Resistance to reinfection varied with the species of Eimeria and with the number of oocysts in the inoculum. Chickens immunized with doses of 20,000 and 80,000 oocysts of E. acewulina, 312 and 1250 oocysts of E. brunetti or E. necatrix, or 1250 and 5000 oocysts of E. maxima at 2 and 4 weeks of age, respectively, were almost completely immune to a challenge dose at 6 weeks of age. Resistance was slightly less in chickens immunized with 1250 and 5000 oocysts of E. aceruulina or 312 and 1250 oocysts of E. maxima. Birds given three immunizing infections of 1250, 5000, and 20,000 oocysts of E. maxima were completeIy immune 8 weeks after the last dose. Resistance was slightly less in birds immunized with similar doses of E. brunetti or E. necatriz. Doses of 20,000, 80,000, and 320,000 oocysts appeared necessary to confer a high level of immunity to E. acervulina. More than three low doses of oocysts appear necessary to induce a complete and enduring immunity against a high challenge for E. acervulina, E. brunetti, and E. necatrix. Higher immunizing doses would not be satisfactory due to the pathogenic effects of the coccidia after the initial infection. INDEX DESCRIPTORS: Eimeria aceroulina; Eimeria brunetti; Eimeria maxima; Eimeria necatrix; Oocysts; Immunity; Chickens.
INTRODUCTION
Dickinson ( 1941), Rose and Long (1962), and Hein ( 1968b) have shown that when repeated coccidial infections are administered to young chickens, a high level of immunity develops. Dickinson (1941) inoculated chickens with E. acervulinu and gave daily doses of oocysts over a period of 5 to 10 days. He showed that there was no change in body weight when birds were heavily challenged 3 to 9 weeks later. Rose and Long (1962) inoculated replicate groups of chickens with two or three rising doses of oocysts of E. acervulina, E. maxima, E. tenella, or E. necatrix, with an interval of 7 days between each infeotion. 250 Copyright
@ 1976
by Academic
Press,
Inc.
All rightr of reproduction in any form reserved.
Their birds were completely protected when challenge’d 38 and 84 days after the initial immunizing infection. Hein (196&b) administered lower doses of E. acervulinu with an interval of 14 days between each infection to replicate groups of 2-week-old chickens and showed that a high level of resistance was conferred against challenge 28 days after the initial infection. Little data are available however on the immunogenic potential of low doses of oocysts, the duration of immunity, or differences in the immunogenicity of different species. Dickinson (1941) and Rose and Long (1962) tended to use high doses of oocysts for immunization with only a short interval of time between each infection and
EIMEFUA
OOCYSTS
TO
IMMUNIZE
their results may have been modified by interference between succeeding infections. Observations on the immunogenic potential of E. maxima appear to be in conflict because the results of Rose and Long ( 1962) do not support the conclusion of Long (1962) that immunity to E. maxima was of short duration. Furthermore, Hein ( 1968b) found that immunity appeared lower in chickens which had received the higher immunizing infections with E. acervulina which was contrary to expectation and requires confirmation. The earlier experiments were carried out under a multiplicity of experimental conditions so that the results are not directly comparable. The present study was undertaken therefore to obtain data on the immunogenic potential of E. acervulina, E. brunetti, E. maxima, and E. necatrix under precisely defined conditions which would provide standards enabling comparisons between species and strains to be made. MATERIALS
Experimental
AND
METHODS
Birds
White Link hybrid cockerel chickens (Sterling Poultry Products Ltd) were used. They were reared in isolation and transferred to the experimental unit 2 days before infection. They were housed under conditions to prevent reinfection and were fed ad libitum on a standard ration (Joyner and Davies 1960). The chickens were e-weeks-old when infected initially (Day 0). Parasitology The oocysts used in the experiments were from the same source as those used previousIy and were prepared under the same standard conditions (Hein 1968a, 1971, 1974). Fresh suspensions of oocysts were prepared for each infection. The age of the oocysts, calculated from the day of recovery from the feces, was 10 or 11 days on each occasion. Dilutions were made to
YOUNG
CHICKENS
251
give the appropriate number of sporulated oocysts ,in a volume of 1 ml on Day 0 for the 2-week-old chickens and in 2 ml on Days 14, 28 and 84 when the chickens were 4-, 6-, ‘and lCweeks-old. After infeotion, the average total daily oocyst output per bird was estimated in a similar manner to that described previously ( Hein 1968a). Experimental
Design
The chickens received either two immunizing doses of oocysts and a challenge infection on Day 28 or three immunizing doses of oocysts and a challenge infection on Day 84. An interval of 14 days was judged to be sufficient to avoid interference between successive infections. The number of oocysts administered in the immunizing and challenge infections are shown in Table I. Low doses of oocysts were chosen for immunization to minimize the detrimental effect on the host and to allow maximum development of the parasite without overcrowding which might modify the immunogenic potential of the coccidia. High doses of oocysts were selected for challenge to produce typical signs of coccidiosis in susceptible chickens. Two days before the initial infection the chickens were weighed and randomized into groups of 10 chickens with approximately equal mean weights. The chickens were weighed daily after each immunizing and challenge infection and total daily oocyst counts were ‘made throughout the course of the experiment. Noninfected chickens served as a control group throughout the experiment. The pathogenicity of the challenge inocula was confirmed in replicate groups of chickens on each occasion. A series of analyses of variance on the daily weights and weight gains of individual birds was carried out, and mean values were compared to assess the statistical significance of the differences between groups.
252
HELEN E. HEIN TABLE: I The Doses of Eimeria spp Oocysts Administered to Young Chickens in the Immunizing and Challenge Infections
Species
E. acervulina
E. brunetti
E. maxima
E.
necutrix
Immunizing
infections
Challenge infections
Day 0
Day 14
Day 28
Day 28
1,250 20,000 320,000 1,250 20,000 320,000
5,000 80,000 1,280,000 5,000 80,000 1,280,000
-
5,120,OOO
312 1,250
1,230 5,000
-
312 1,250
1,250 5,000
5,000 20,000
312 1,250
1,250 5,000
-
1,250 20,000
5,000 80,000
20,000 320,000
1,250 312
5,000 1,250
- 5,000
1,250
5,000
20,000
20,000 320,000 6,120,000
Day 84
10,240,OOO
320,000 i 1,28O,OC^ I” 320,000 1,280,000 320,000 >
320,000
equalled that of the noninfected controls and by Day 26 there was no significant The effects of the immunizing infections difference in mean weight between these are shown in Table II and Figs. la, lb, lc, and Id, and those of the challenge infec- and the birds infected with the two lower tions in Tables III and IV and Figs. 2a, 2b, doses of oocysts (Fig. la). However, the 2c, 2d and 3a, 3b, 3c, and 3d. mean weight of the birds inoculated with the highest doses of oocysts was signifiE. acervulina cantly lower (P
EIMERL4
OOCYSTS
TO
IMMUNIZE
infection was the presence of oocysts in the feces during the patent phase of the disease. In birds immunized with the lowest doses of oocysts, total output was approximately 2 and 77 million oocysts per bird, respectively, whilst in the chickens immunized with either of the higher doses of oocysts the number was too low to count (Tables III and IV). E. brunetti After the first infection, morbidity and retardation of growth were marked on Days 5 and 6, ,the effect being most severe in the group receiving the higher dose of 1250 oocysts in which one bird died on
YOUNG
Day 6 (Table II, Fig. lb). The mean weight gains were significantly lower in the infected chickens than in the noninfected controls ( P
TABLE The Infection
Pathogenic
Effects
1
2
II
of the Immunizing 3
1
253
CHICKENS
Infections 2
with 3
Eimeria
spp
1
2
3
320,000 +” -
1,280,OOO -
5,120,OOO -
+
+
E. acervulina Dose Clinical signsa Mortality Mean y0 wt lossa Oocysts~
1,250 4 19
5,000 -
+”
20,000 -
20,000 -
+
320,000 -
1,250 ++ l/10 22 20
5,000 -
20,000 -
1,250 -
5,000 -
20,000 -
8 73
80,000 -
19 30
E. brunetti Dose Clinical signs Mortality Mean y0 wt loss oocysts
312 +i-
5,000 -
14 20
E. maxima Dose Clinical signs Mortality Mean $& wt loss Oocysts
312 10 3
1,250 -
-
11 7
20,000 + 18 5
80,000 -
320,000 -
E. necatrix Dose Clinical signs Mortality Mean y0 wt loss Oocysts
312 -
1,250
-
10
a Clinical signs : + denotes slight b Mean percentage loss in weight 0 Average total oocyst production too small to count.
5,000 -
1,250 12
5,000 -
20,000 -
anorexia, + + marked anorexia during immunization compared per bird in millions, + denotes
and depression. with noninfected control birds. oocysts found on salt flotation,
number
254
HELEN
A
E.ACERVUlINA
E. HEIN
E .BRUNETTI
lb
E MAXIMA
DAYS AFTER INOCULATION FIG. 1. The mean weight in grams of chickens after inoculation with the first and second immunizing doses of Eimeriaspp. oocysts. Key: -ononinfected chickens; -n312 + 1250 oocysts; -x-1250 + 5000 oocysts; -A-20,000 + 80,000 oocysts; -W-320,000 + 1,280,OOO oocysts.
2 days before challenge on Day 82. Following challenge on Day 28 or Day 84, there was no indication of morbidity or growth retardation in the immunized chickens (Tables III and IV, Figs. 2b and 3b). A few oocysts were detected in the feces of the immunized birds but the number was too low to count (Tables III and IV). E. maxima After the first infection, slight retardation of growth was noted on Day 7 in the chick-
ens inoculated with 312 oocysts (P
EIMERIA
OOCYSTS
TO
IMMUNIZE
with 1250 or 20,000 oocysts was slightly lower than that of the noninfected birds (P
Pathogenic
Effects
of Eimeria
255
CHICKENS
mean weight between the immunized birds and the noninfected chickens were not significant. E. necatrix After the first infection, there was no sign of morbidity or retardation of growth in either group of chickens. The average total production of oocysts was 10 and 12 million oocysts per bird, respectively (Table II). After the second and third infections, no oocysts were detected in the feces. Following challenge on Day 28, there was no evidence of infection in either group of immunized chickens (Table III, Fig. 2d). After challenge on Day 84, no sign of morbidity or growth retardation was noted in the immunized chickens, a few oocysts were detected in the feces of each group but the number was too small to count (Table IV, Fig. 3d). The pathogenicity of the challenge infections on Day 28 and Day 84 was con-
TABLE The
YOUNG
III
spp. Challenge
Infections
Administered
on Day
E. acervulina Dose Immunizing dose Clinical signs” Mortality Mean To weight oocysts x 106
None ++” lossb
8 75
5,120,OOO
1,250 5,000 -
E. brunetti oocysts
20,000 80,000 -
-
+”
2
Dose 320,000 1,280,OOO +
None +++” 30 10
E. maxiwra Dose Immunizing dose Clinical signs Mortality Mean y0 weight) Oocysts x 106
None +++ loss
21 6
320,000
28
320,000
oocysts
312 1,250 -
1,250 5,000 -
+
+
E. necatrix oocysts
312 1,250 -
1,250 5,000 -
+
-
Dose None ++++” lO/lO
@Clinical signs : + + marked anorexia and depression : + + + marked anorexia, stained feces : + + + + very high morbidity. 6 Mean ‘% weight loss after challenge compared with noninfected control chickens. c + denotes a few oocysts found on salt flotation, number too small to count.
320,000
oocysts
312 1,250 depression
1,250 5,000 and blood
256
HELEN
E. HFJN
TABLE
IV
The Pathogenic Effects of Eimeria spp. Challenge Infections Administered E.
acervulina
E. brunetti
Dose 10,240,OOO oocysts Immunizing doses
None
1,250 5,000 20,000
Clinical signs Mortality Mean y0 weight lossa oocysts x 106
+++” l/8
320,000
7
-
Dose 1,280,OOO oocysts 320,000
77
$6
None
5,120,000
-
T
5,000
+++ 4/8 26 44
E. maxima
Dose 1,280,OOO oocysts Immunizing doses
None
Clinical signs Mortality Mean ‘% weight loss oocysts x 106
+++ 16
1,250 5,000 20,000
20
20,000
E.
None
320,000
-
-
-
1,250 5,000 20,000
-
-
-
-
+
+
-
necatrix
Dose 320,000 oocysts
80,000
-
312 1,250
1,280,000
-
-
192
20,000
80,000
on Day 84
++++” lO/lO
312 1,250
1,250 5,000
5,000
20,000
-
-
+
+
a Mean y0 weight loss after challenge compared with noninfect,ed control chickens. b + denotes a few oocysts found on salt flotation, number too small to count. c Clinical signs : + + $ High morbidity : + + + 4 Very high morbidity.
feces of the immunized chickens after challenge on Day 84. Chickens inoculated with E. brunetti passed a few oocysts after each challenge infection suggesting that resistance was slightly lower in these birds. DISCUSSION With E. acervulina, approximately 2 milA very high level of resistance to reinfeclion oocysts per bird were produced after tion was conferred by multiple infections challenge on Day 28 which suggested that with relatively low doses of oocysts of E. the immunogenic potential of this species acervulina, E. brunetti, E. maxima, or E. was lower. This was confirmed by the relanecatrix. tively high output of 77 million oocysts Small differences were apparent in the after challenge on Day 84. immunogenic potential of these species of With all four species, even the lowest coccidia. In chickens immunized with stan- doses of oocysts produced an easily demdard doses of 1250 and 5000 oocysts and onstrated immunity. There was no clinical challenged on Day 28, or given a third dose response to any of the challenge infections. of 20,000 oocysts and challenged on Day However, in chickens immunized with the 84, no evidence of infection was detected smallest doses of E. maxima a few oocysts in the birds inoculated with E. maxima were detected in their feces after chalwhich indicated that these birds were com- lenge on Day 28, while in birds inoculated pletely immune. Resistance was also very with E. acervulina, oocyst production was high in birds receiving E. necatrix, al- demonstrably greater when they had rethough a few oocysts were present in the ceived the lowest immunizing doses, The firmed by the typical signs of acute coccidiosis with h6gh mortality in each group of susceptible chickens (Tables III and IV, Figs. 2 and 3).
ElIMERIA
OOCYSTS
TO
IMMUNIZE
results for these two species suggest that the level of immunity is influenced by the number of oocysts administered. These observations are in general agreement with ‘those of Rose and Long (1962) who reported that the immunizing activity of coccidia varied with the species of Eimeria, and with the work of Rose (1971) which indicated that in the case of E. maxima protection was dose dependent. Differences in the effects of the immunizing and challenge infections showed that
YOUNG
CHICKENS
257
the optimum number of oocysts for immunization varied with the species of Eimeria. When chickens received two immunizing infections followed by a challenge infection on Day 28, the reduced oocyst production after challenge in birds infected with 20,000 and 80,000 oocysts of E. acervulina indicated that these doses of oocysts will not give total immunity but will give protection against the clinical effects of a high challenge infection. The high mortality and marked weight loss associated *co
I
I
E BRUNETTI
2b
DAYS AFTER CHALLENGE FIG. 2. The mean weight in grams of immunized and susceptible chickens after Eimetia spp. challenge on Day 28. Key: -@-noninfected chickens; -Asusceptible chickens; -nimmunized 312 + 1250 oocysts; -Ximmunized 1250 + 5000 oocysts; -Aimmunized 20,000 + 80,000 oocysts; ---320,000 + 1,250,OOO oocysts.
258
HELEN
E.
HEIN xmo
?mw
E.BRUNETTI
E.ACERVULINA
2000
E .MAXIMA
E. NECATRIX
DAYS AFTER CHALLENGE FIG. 3. The mean weight in spp. challenge on Day 84. Key: immunized 312 + 1250 + 5000 immunized 20,000 + 80,000 + 5,120,OOO oocysts.
grams of immunized and susceptible chickens after Eimeria -o-- noninfected chickens; -Asusceptible chickens; -ooocysts; -Ximmunized 1250 + 5000 + 20,000 oocysts; --A320,000 oocysts; -H-immunized 320,000 + 1,280,OOO +
with infections of E. brunetti (Hein 1974) suggests that a dose of 312 followed by 1250 oocysts approaches the maximum tolerated by young chickens. The presence of a few oocysts after challenge in birds inoculated with similar numbers of oocysts of E. maxima indicated that the higher doses of 1250 and 5000 oocysts are necessary to obtain complete immunity. Both regimes used for E. necatrix gave good immunity against both challenge infections without obvious clinical effects in the birds. However, in view of the high pathogenicity
of this species of coccidia (Hein 1971) and the difference in susceptibility of chickens to infection associated with breed (Long 1968) it would seem wise to select the lower doses of 312 and 1250 oocysts for immunization. The results from groups given three immunizing infections and challenged on Day 84 were complementary to those in which birds received 2 immunizing doses of oocysts and a challenge on Day 28. However the experiments were not strictly comparable, so that further studies are necessary to establish the mini-
EIMEFUA
OOCYSTS
TO IMMUNIZE
mum number of doses of oocysts required to confer a high and enduring immunity. The virtual absence of oocysts in the droppings after the second immunizing infection suggests that a high level of immunity may arise from the initial infection and recent studies by Rose (1971) have indicated that with E. maxima this protection may be at its greatest on the 14th day after infection. This coincides with the time of the second immunizing dose of oocysts in the present study. In the experiments recorded by Rose and Long ( I962), high numbers of oocysts were produced in the feces of the birds after the second immunizing infection, and comparisons of the immunogenic potential of each species were based upon the observed oocysts production. The differences between their observations and those in the present experiments in which very little oocyst production was seen after the second infection, may have been due to the particular doses used and to the shorter time interval of 7 days which Rose and Long allowed between successive infections. Pout (1969) studied the changes in the intestinal mucosa following infection with Eimeria sp. and suggested that at least 14 days must elapse before the tissue regains its normal architecture; infections given within this intervaI may be modified by tissue changes. The level of immunity conferred by doses of 20,000 and 80,000 oocysts of E. acervuha appeared similar to that in birds inoculated with 80,000 and 160,000 oocysts of the same species in earlier studies (Hein 196813). Immunity was almost complete in birds infected with the highest doses of 320,000 and 1,280,OOO oocysts throwing considerable doubt on the previous results in which resistance appeared to decrease when the immunizing infections were increased from 80,000 and 160,000 to 320,000 and 640,000 oocysts (Hein 196813). Resistance in birds immunized with doses of 20,000, 80,000, and 320,000 oocysts of
YOUNG
CHICKENS
259
appeared comparable to that in birds given repeated daily infections with approximately the same total number of oocysts by Dickinson ( 1941). The presence of a few oocysts in the feces of 1the immunized birds after challenge indicated that immunity was lower than that obtained by Rose and Long (1962) with far higher doses of 500,000, 5,000,000, and 10,000,000 oocysts, when no evidence of infection was detected after challenge with 20,000,000 oocysts on both Day 38 and Day 84. Changes in body weight were not mentioned by these workers but dose response studies (Hein 1968a) suggest that the detrimental effects of the parasite would have been severe after the initial immunizing infection. This receives support from the marked growth retardation in birds inoculated with 320,000 oocysts in the current study. Thus it would seem preferable to employ low doses of oocysts for immunization ‘and to increase the number of infections to induce a high level of immunity and avoid growth retardation. The presence of oocysts in the feces of birds after challenge on Day 84 in birds immunized with oocysts of E. brunetti or E. necatrix suggests that with these species, more than three infections are required to induce a lasting complete immunity against a high challenge infection. Rose and Long ( 1962) immunized birds with doses of 500, 5000, and 50,000 oocysts of E. necatrix and found no evidence of infection when the same group of birds was challenged with 100,000 oocysts both on Day 38 and Day 84. Their birds had received, in effect, four immunizing infections at the time of the final challenge on Day 84. In the present experiment, immunizing infections were far lower, the challenge infection was higher and the time interval between immunization and challenge was greater. The results suggest that the large doses of E. necatrix used by Rose and Long (1962) are not necessary to produce an effective immunity to this species. E. acetwlina
260
HELEN
The high level of immunity in birds inoculated with E. maxima confirmed the observations of Rose and Long (1962) that this species has a high immunogenic potential. The results however throw considerable doubt on the observation of Long (1962) that immunity to E. maxima is of short duration. ACKNOWLEDGMENTS The author wishes to thank Dr. L. P. Joyner for help in the preparation of the paper and Miss N. Hebert for her statistical analysis of the results. She also thanks Mr. R. Sayer and Mr. P. Hall for their help in the preparation of Figs. 1-3 and Mr. C. Norton and Mr. P. Hall for their excellent technical assistance. REFERENCES DICKINSON, E. M. 1941. The effects of variable dosages of sporulated Eimeria acertiulina oocysts on chickens. Poultry Science 20, 413-424. Hum, H. E. 1968a. The pathogenic effects of Eimeria acervulina in young chicks. Experimental Parasitology 22, l-11. HEIN, H. E. 196813. Resistance in young chicks to
E. HEIN reinfection by immunization with two doses of oocysts of Eimeria acewulina. Experimental Parasitology 22, 12-18. HEIN, H. E. 1971. Pathogenic effects of Eimeria necatrix in young chickens. Experimental Parasitology 30, 321330. HEIN, H. E. 1974. The pathogenic effects of Eimeria brunetti in young chickens. Experimental Parasitology 36, 333-341. JOYNER, L. P., AND DAVIES, S. F. M. 1960. Detection and assessment of sublethal infection of Eimeria tenella and Eimeria necatrix. Experimental Parasitology 9, 243-249. LONG, P. L. 1962. Observations on the duration of acquired immunity of chickens to Eimeria maxima Tyzzer, 1929. Parasitology 52, 89-93. LONG, I’. L. 1968. The effect of breed of chickens on resistance to Eimeria infections. British Podtry Science 9, 71-78. POUT, D. D. 1969. Observations on coccidiosis of grazing lambs with special reference to the mucosal reaction of the small intestine. Ph.D. thesis, University of London. ROSE, M. E., AND LONG, P. L. 1962. Immunity to four species of Eimeria in fowls. Immunology 5, 79-92. ROSE, M. E. 1971. Immunity to coccidiosis: protective effect of transferred serum in Eimeria maxima infections. Parasitokgy 62, 11-25.