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Eimeria brunetti: Cross infections in chickens immunized to E. maxima
ISSpERIMEN'I'.~L
Eimeria
PARASI'TOLOGY
brunetti:
29, 367-374 (1971)
Cross
Infections
in Chickens Helen
Central
Veterinary
Laboratory, Ministry Yeu, Haw, Weybridge, (Submitted
Immunized
to E. maxima
Hein of Agriodture, Fisheries Surrey, England
for publication,
3 March
rind Food,
1970)
HEIR’, HELEN. 1971. Eimeria brunetti: Cross infections in chickens immunized to E. maxima. Experimentrrl Parasitology 29, 367-374. Chickens immunized with three doses of oocysts of Eimeria mnxima (Protozoa; Sporozoa) were completely protected against reinfection with high challenge doses of E. maxima but mere completely susceptible to cross infection with low or high doses of oocysts of E. brwetti. Chickens immunized with different numbers of oocysts of E. maxima behaved in a similar manner. There was no evidence of cross immunity between the two species. INDEX
Eimeria
Coccidiosis; Protozoa.
DESCRIPTORS:
maxima;
Cross infections;
Since the early work of Tyzzer, Theiler, and Jones (1932 j it has been accepted that resistance to reinfection with Eimeria sp. (Protozoa, Sporozoaj is species specific and this fact has been accepted as the basis of much subsequent work on immunology and species identification. Recently, doubt was thrown on the belief that immunity is species specific when Rose (1967) suggested a possible immunological relationship between E. brunetti and E. maxima. Chickens immunized with one species appeared resistant to cross infection with the other. The specificity of immunity is of fundamental importance in both field and laboratory studies on coccidiosis, therefore further investigation of the effects of cross infection with E. brunetti and E. m.axima was necessary. Preliminary experiments using the Weybridge strain of E. brunetti and E. maxima failed to show any indication of cross immunity between these species. Therefore an experiment was designed with the Houghton strains of E. brunetti and E. maxima, as used by Rose in 1967, in case the different results at Weybridge might be attributable to strain differences. The doses of oocysts 367
Immunity;
Eimeria
bwnetti;
selected for immunization, challenge, and cross infection were based on the results of earlier work with these species at Weybridge. Low doses of oocysts were selected for the final cross infection to ensure that if the level of cross immunity was low it would not be masked by “over challenge.” Further replicate groups of chickens were immunized, challenged, and cross infected in a similar manner to that described by Rose (1967) in case the results might be influenced by the doses of oocysts used for immunization or challenge. The results of the current experiments are recorded in detail due to the complete lack of agreement with those of Rose (1967). MATERIALS
AND METHODS
Experimental birds. White link hybrid cockerel chickens (Sterling) were used in the study. The chickens were reared in isolation and transferred to the experimental unit 2 days before infection. The chickens were fed ad libitum on a standard ration (Joyner and Davies 1960). The cages were cleaned daily during the patent phase of the disease to avoid reinfection. The chickens
368
HEIN
TABLE
I
The Doses of Ooc~.sts of Eimeria maxima und R. brunetti Chickens in Experimenis 1, 2 and 3
Administered
lo the
-
Doses of Oocysts in Thousands;
Group:
Day Experiment la 0 Infection with E. maxima 14 28 42 42 Infection with E. brunetti
3
5
-
1.25 5 20 320
1.25 5 20
-
--
320
-
1.25
Experiment 2a 0 Infection with E. maxima 14 28 42 56 56 Infection with E. briLnetli
320
1.25 5 20 320 320
-
-
6
1.25 5 20
1.25
20
20
1.25 5 20 320 1.25
Experiment 3b 0 Infection with E. maxima 14 28 42 42 Infection with E. brunelli a Chickens 6 Chickens
2
1.25 5 20 320
1.25
20
20
5 50 100
50
-
-
50
50
-
-
2 weeks old on day 0. 4 weeks old on day 0.
n 0
8
i4
12
0
DAYS
FIG. 1. The mean weight maxima.
of the chickens after the first immunizing
+4 DAYS
a
12
doses of oocysts of E.
E. brunetti were 2 or 4 weeks old on the day of the first immunizing infection. Parasitology. The oocysts used in this experiment were derived from the Houghton strains of E. maxima and E. brunetti. Fresh suspensions of oocysts were prepared for each infection. The age of the oocysts calculated from the day of recovery from the feces was 9 or 10 days on each occasion. Approximately 70% of the oocysts were sporulated. Dilutions were made to give the appropriate number of sporulated oocysts in a volume of 1 ml on day 0 and in a volume of 2 mi when the birds were infected on day 14,28,42, and 56. The average total daily output of oocysts per bird was estimated in a MeMaster counting chamber in a manner similar to that described earlier (Hein 1968). Fecal ramples were examined by salt flotation when the number of oocyst,s was t’oo small to count in a Mchlastcr slide. Experimental design. Two days before infect,ion the chickens were weighed and divided at random into groups of 10 birds. The number and species of oocysts administered to the chickens are shown in Table I. Three methods of inducing immunity were adopted. In Experiments 1 and 2, replicate groups of chickens (groups 3, 5, and 7) were infected with low doses of 1,250, 5,000, and 20,000 oocysts of E. maxima with an interval of 14 days between each infection. In Experiment 2, to ensure that immunity to E. maxima was complete, the chickens received a fourt,h higher dose of 320,000 oocysts, 14 days after the third infection. In Experiment 3, t’he chickens were immunized by the method described by Rose (1967). Fourteen days after the last immunizing infection on day 42 and day 56 in Experiments 1 and 2, respectively, the chickens in group 3 received a standard challenge dose of 320,000 oocysts. On the same day replicate groups of immunized chickens (groups 5 and 7) received either a dose of 1,250 or
369 TABLE
II
The Average Total Daily 0ociJ.d l’ro-lt,clion per Bird, Calrulaterl to the Searest Million Uocysts, During Immunization with OOC~:~JS~.S of Eimeria maxima (Houghton Strwin)
Day
Experiments 1 and 2; Groups 3, 5, and 7 ____~___ Oocysts Oocyst inocupoduclated tion
0 r; 7 8 0 10 11 12 13
1250
14
5000
Exrxriment 3; Groups 3 and 5 Oocysts Oocyst inocu- produclated tion 5000 2.0 18.0 0.0 + + + + + 26 -
+” 6.0 3.0 + + + + -9 50000 Xi1
28
20000
Nil 100000
Xi1 34 35 36 37 n + denotes oocysts detected number too small to count.
+ + + + on salt flotation,
20,000 oocysts of E. brunetti. In Experiment 3, on day 42 the chickens in group 3 received a challenge dose of 50,000 oocysts of E. maxima and those in group 5 received a dose of 50,000 oocysts of E. brunetti in accordance with the method of Rose (1967). The pathogenicity of the challenge and cross infections were confirmed by inoculation of susceptible birds in groups 2, 4, and 6 on day 42 or day 56 as indicated in Table I. The chickens were weighed regularly t’hroughout the experiments. Clinical signs and mortality were recorded during the acute phase of each infection and total oocyst counts were made during the patent period of the disease.
370
HEIN
TABLE l’he Average
III
l’ofal Daily Oocyt Production per Bird, Calculated to the Nearest Million Ooc!/sts, Reinfecfion wi/h, Eimeria maxima or Cross Tnfeclion with Xmerin hrlmetti Susceptible chickens
Experiments
Group : species: dose :
2 E. maxima
4 R. brunetti
E. maxima-immunized
I-
3 I? R. maxima brunetti
After
chickens
5 E. brwzetti
7 R. , brunetti
-
J20,OOO1 51DO,00 1,250 __ -~
50,000
J20,OOO 510,000
,-a
l)ny
5 0 7 8 9 10 Total 5 0 7 8 9 10 Tot al
3.3 18.0 1.7 0.1 + 23
8.0 14.2 2.0 0.4 0.1 25
14.2 11.2 3.5 0.4 + 29
0.4 17.3 4.7 0.4 0.2 23
22.0 27.0 3.5 1.1 0.3 54
+ 13.2 7 (i 1.8 0.3 0.1 23
Ilay
l)ay
20,000
-
16.8 13.8 2.7 0.8 + 34
~
14.0 7.1 1.8 0.2 + 23
+ 14.4 9.4 2 (i 1.1 0.2 28
18.5 5.0 1.8 0.4 0.1 26
-
5
10 Totztl
2ti. 0 9.0 3.0 1.2 i 0.1 ~ 39
19.0 5.7 2.9 0.2 + 28
0.5 16.0 4.0 0.2 0.2 21
(i 7 8 9
a + oocysts
detected
on salt flotittion,
number
too small to comet.
RESULTS
Effects of the immunizing infections. After the first infection no sign of anorexia or depression was noted although growth was slightly retarded between day 5 and day 7 (Fig. 1) and oocysts were produced during the patent phase of the infection (Table II) . After the second and third infections no detrimental effects on growth were detected, although the mean weight of the immunized chickens was still, but not significantly, less than that of the noninfected chickens. Oocysts were not detected in the feces of
the chickens after the second infection. A few oocysts were present in the feces of the chickens given the highest dose in the third infection, between day 34 and day 37 in Experiment 3 (Table II). Effects of the challenge infections with E. maxima. After reinfection with the high challenge doses of oocysts on day 42 (Experiments 1 and 3) or day 56 (Experiment 2) no evidence of infection was detected in the immunized chickens in group 3. Typical signs of coccidiosis, however, were seen in the previously uninfected chickens in group 2 (Tables III and IV, Figs. 2 and 3). Effects of the cross infection with E. bru-
E. brunetti TABLE
371
I\’
R. nza.~ime-immunized chickens
Susceptible chickens Esperiment
Group : species: dose:
R.
brunetti ~0,000
1
2
Clinical + signs Mortality Cl0Weight lossc oocyst x ; 10” ~ Clinical signs I 1lortalitJ 67 ,o Weight loss oocq-sts 106
1,250 ++++I
+‘I
I,‘10 13
~ 3 23
I-t+++’
++
++++ ‘.‘VI0 13
1 ‘10 16 23
Ii -+++
/++++I ~
2 10 ‘1
1
a ++ marked anorexia atld depression. b ++++ complete anorexia, very high morbidity. c The mttxim~m~ difference it1 weight betwren the immunized or slwceptible chickells atid the llonix,fected chickens on the 6th or 7th day after challenge or cross infection expressed as a percentage of the weight of the rloninfected chickells.
netti. In all three experiments typical signs of acuic coccidiosis occurred in both the susceptible control chickens (groups 4 and 6) and the chickens immunized to E. maxima (groups 5 and, 7) after infection with low or high doses of oocysts of E. brunetti. There was no significant difference between the severity of infection in the susceptible control chickens and t.he immunized ehickens given similar doses of oocysts of E. brunetti. The pathogenic effects of E. brunetti appeared similar in chickens immunized ei-
ther in an identical manner t’o that used by Rose in 1967 (Experiment 3) or with doses of oocysts of E. maxima as used at Weybridge (Experiments 1 and 2). Morbidity was high, deaths ranged from 10 to 40% and characteristic lesions of hemorrhagic enteritis were found in birds which died. On day 5 and 6 cyanosis was marked, anorexia was complete, and the birds tended to sit on the pen floor with quivering tail feathers: dysentery was severe. The mean weight fell on the 5th, 6th, and 7th days after infection
372
HEIN
KEY DOSE OF OOCYSTS x16 42
42
NIL
NIL
NIL I
NIL
.-a NIL NIL O-0 1.25 5
NIL 20
320 320
c-a ‘NIL
c-2
E.BRUNETTI
E.MAXIMA 14 28
DAY0
NIL NIL
x-x
1.25 5
M
125
x-*
1.25 5
20
20
0
NIL NIL w. NIL
1.25
Y .-.
+2
20
10
12
FIG. 2. The mean weight of the chickens after challenge with oocysts of E. mtima cross infection with oocysts of E. brunetti (Experiment 1).
and was still lower than the noninfected chickens on the 12th day (Figs. 2 and 3). After adjustment by covariance to allow for differences in mean weight on the 3rd day, the mean weights of the infected chickens
or
were significantly lower than the noninfected birds on the 7th day (P = 0.001) and the 12th day (P = 0.01). Small differences in weight were apparent between certain groups after infection with E. brunetti
373
E. brunetti KEY
but they were not significant. Total oocyst production varied slightly between groups receiving the lowest dose of 1,250 oocysts of E. brmetti but there was little difference in oocyst output of chickens receiving the higher challenge doses of 20,000 and 50,000 oocysts of E. brunetti (Table III).
DOSEOFOCCYST5XlO’
I4
28
42
42 NIL
w
NIL
NIL
NIL
NIL
. .
NIL
NIL
NIL
50
ao5 AA
50xX) NIL
x-x 5
DISCCSSION The results showed conclusively that chickens immunized with oocysts of E. maxima were fully prot’ected against a high challenge infection wit’h the same species but that they were completely susceptible to cross infection with low or high doses of oocysts of E. brunetti. The results were similar in chickens immunized, challenged, and cross infected with different numbers of oocysts. The results of the present study using the Houghton strains of E. brunetti and E. maxima were identical with those of earlier work using the Weybridge strains of these species. There was no evidence of cross immunity (Tables III and IV) and these findings therefore support the observations of Tyzzer, Theiler, and Jones (1932) who considered that resistance to reinfection with Eimeria sp. was species specific. These results directly disagree with the findings of Rose (1967). Analysis of the data reported by Rose (1967) shows that large numbers of oocysts were passed after both the first and the second immunizing infections. This result differs significantly from that of Rose and Long (1962) in which oocyst production was negligible after the second immunizing infection with E. maxima and from current immunological studies at Weybridge employing multiple infections in which oocyst production was also minimal after the second and third immunizing infections with either E. acervulina, E. brunetti, E. maxim.a, or E. necatrix. The discrepancies connot be due to strain variation between the Houghton and Weybridge strains of E. maxima and E. brunetti, nor is it likely
E.BRUNETTI
EMAXIMA
MY0
NIL
50 50
NH
50
50 KM
p”
c-2
0
+2
4 au5
6
rp
8
10
FIG. 3. The mean weight of chickens after challenge with oocysts of E. maxima or cross infection with oocysts of E. brunetti (Experiment 3).
that differences in experimental diet or breed of bird could be responsible for such a marked difference in oocyst production. Howercr, the high product’ion of oocysts
374
HEIN
after the second immunizing infection in Rose’s experiment could be an indication of an adventitious infection at this time, which, if it were E. brunetti, would explain the apparent immunological relationship between E. brunetti and E. maxima and the complete failure in the present study using the Houghton strains of Eimeria to confirm the work of Rose (1967). ACKKOWLEDGMENTS Acknowledgments are due to Dr. M. E. Rose who kindly supplied suspensions of oocysts of the Houghton strains of E. brunetti and E. mnximn. The author thanks Mr. S. F. M. Davies and Dr. L. P. Joyner for their helpful discussions during this study and Miss N. Hebert for her statistical analysis of the results. I acknowledge the cooperation of Mr. R. Sayer in the preparation of Figs.
l-3 and thank Mr. C. Norton and Mr. P. Hall for their invaluable technical assistance. REFERENCES HEIK, H. E. 1968. The pathogenic effects of Eimeria aceruuliw in young chicks. Experimental Parasitology 22, l-11. JOYS-ER,L. P., .~NDDAVIES. S. F. M. 1960. Detection and assessment of sublethal infections of Eimeria tenella and Eimeria necatrix. Experimental Parasitology 9,243-249. ROSE, M. E. 1967. Immunity to Eimeria hnetti and Eimeria maxima infections in the fowl. Parasitology 57,363-370. ROSE, M. E., AND LOKG. P. L. 1962. Immunity to four species of Eimeria in fo\&. Immunology 5,79-92. TYZZER,E. E.. THEILER, H., ASD Jo~ves.E. E. 1932. Coccidiosis of gallinaceous birds. II. A comparative study of species of Eimerin of tht: chicken. American Journal of Hygiene 15,319393.
Eimeria
PARASI'TOLOGY
brunetti:
29, 367-374 (1971)
Cross
Infections
in Chickens Helen
Central
Veterinary
Laboratory, Ministry Yeu, Haw, Weybridge, (Submitted
Immunized
to E. maxima
Hein of Agriodture, Fisheries Surrey, England
for publication,
3 March
rind Food,
1970)
HEIR’, HELEN. 1971. Eimeria brunetti: Cross infections in chickens immunized to E. maxima. Experimentrrl Parasitology 29, 367-374. Chickens immunized with three doses of oocysts of Eimeria mnxima (Protozoa; Sporozoa) were completely protected against reinfection with high challenge doses of E. maxima but mere completely susceptible to cross infection with low or high doses of oocysts of E. brwetti. Chickens immunized with different numbers of oocysts of E. maxima behaved in a similar manner. There was no evidence of cross immunity between the two species. INDEX
Eimeria
Coccidiosis; Protozoa.
DESCRIPTORS:
maxima;
Cross infections;
Since the early work of Tyzzer, Theiler, and Jones (1932 j it has been accepted that resistance to reinfection with Eimeria sp. (Protozoa, Sporozoaj is species specific and this fact has been accepted as the basis of much subsequent work on immunology and species identification. Recently, doubt was thrown on the belief that immunity is species specific when Rose (1967) suggested a possible immunological relationship between E. brunetti and E. maxima. Chickens immunized with one species appeared resistant to cross infection with the other. The specificity of immunity is of fundamental importance in both field and laboratory studies on coccidiosis, therefore further investigation of the effects of cross infection with E. brunetti and E. m.axima was necessary. Preliminary experiments using the Weybridge strain of E. brunetti and E. maxima failed to show any indication of cross immunity between these species. Therefore an experiment was designed with the Houghton strains of E. brunetti and E. maxima, as used by Rose in 1967, in case the different results at Weybridge might be attributable to strain differences. The doses of oocysts 367
Immunity;
Eimeria
bwnetti;
selected for immunization, challenge, and cross infection were based on the results of earlier work with these species at Weybridge. Low doses of oocysts were selected for the final cross infection to ensure that if the level of cross immunity was low it would not be masked by “over challenge.” Further replicate groups of chickens were immunized, challenged, and cross infected in a similar manner to that described by Rose (1967) in case the results might be influenced by the doses of oocysts used for immunization or challenge. The results of the current experiments are recorded in detail due to the complete lack of agreement with those of Rose (1967). MATERIALS
AND METHODS
Experimental birds. White link hybrid cockerel chickens (Sterling) were used in the study. The chickens were reared in isolation and transferred to the experimental unit 2 days before infection. The chickens were fed ad libitum on a standard ration (Joyner and Davies 1960). The cages were cleaned daily during the patent phase of the disease to avoid reinfection. The chickens
368
HEIN
TABLE
I
The Doses of Ooc~.sts of Eimeria maxima und R. brunetti Chickens in Experimenis 1, 2 and 3
Administered
lo the
-
Doses of Oocysts in Thousands;
Group:
Day Experiment la 0 Infection with E. maxima 14 28 42 42 Infection with E. brunetti
3
5
-
1.25 5 20 320
1.25 5 20
-
--
320
-
1.25
Experiment 2a 0 Infection with E. maxima 14 28 42 56 56 Infection with E. briLnetli
320
1.25 5 20 320 320
-
-
6
1.25 5 20
1.25
20
20
1.25 5 20 320 1.25
Experiment 3b 0 Infection with E. maxima 14 28 42 42 Infection with E. brunelli a Chickens 6 Chickens
2
1.25 5 20 320
1.25
20
20
5 50 100
50
-
-
50
50
-
-
2 weeks old on day 0. 4 weeks old on day 0.
n 0
8
i4
12
0
DAYS
FIG. 1. The mean weight maxima.
of the chickens after the first immunizing
+4 DAYS
a
12
doses of oocysts of E.
E. brunetti were 2 or 4 weeks old on the day of the first immunizing infection. Parasitology. The oocysts used in this experiment were derived from the Houghton strains of E. maxima and E. brunetti. Fresh suspensions of oocysts were prepared for each infection. The age of the oocysts calculated from the day of recovery from the feces was 9 or 10 days on each occasion. Approximately 70% of the oocysts were sporulated. Dilutions were made to give the appropriate number of sporulated oocysts in a volume of 1 ml on day 0 and in a volume of 2 mi when the birds were infected on day 14,28,42, and 56. The average total daily output of oocysts per bird was estimated in a MeMaster counting chamber in a manner similar to that described earlier (Hein 1968). Fecal ramples were examined by salt flotation when the number of oocyst,s was t’oo small to count in a Mchlastcr slide. Experimental design. Two days before infect,ion the chickens were weighed and divided at random into groups of 10 birds. The number and species of oocysts administered to the chickens are shown in Table I. Three methods of inducing immunity were adopted. In Experiments 1 and 2, replicate groups of chickens (groups 3, 5, and 7) were infected with low doses of 1,250, 5,000, and 20,000 oocysts of E. maxima with an interval of 14 days between each infection. In Experiment 2, to ensure that immunity to E. maxima was complete, the chickens received a fourt,h higher dose of 320,000 oocysts, 14 days after the third infection. In Experiment 3, t’he chickens were immunized by the method described by Rose (1967). Fourteen days after the last immunizing infection on day 42 and day 56 in Experiments 1 and 2, respectively, the chickens in group 3 received a standard challenge dose of 320,000 oocysts. On the same day replicate groups of immunized chickens (groups 5 and 7) received either a dose of 1,250 or
369 TABLE
II
The Average Total Daily 0ociJ.d l’ro-lt,clion per Bird, Calrulaterl to the Searest Million Uocysts, During Immunization with OOC~:~JS~.S of Eimeria maxima (Houghton Strwin)
Day
Experiments 1 and 2; Groups 3, 5, and 7 ____~___ Oocysts Oocyst inocupoduclated tion
0 r; 7 8 0 10 11 12 13
1250
14
5000
Exrxriment 3; Groups 3 and 5 Oocysts Oocyst inocu- produclated tion 5000 2.0 18.0 0.0 + + + + + 26 -
+” 6.0 3.0 + + + + -9 50000 Xi1
28
20000
Nil 100000
Xi1 34 35 36 37 n + denotes oocysts detected number too small to count.
+ + + + on salt flotation,
20,000 oocysts of E. brunetti. In Experiment 3, on day 42 the chickens in group 3 received a challenge dose of 50,000 oocysts of E. maxima and those in group 5 received a dose of 50,000 oocysts of E. brunetti in accordance with the method of Rose (1967). The pathogenicity of the challenge and cross infections were confirmed by inoculation of susceptible birds in groups 2, 4, and 6 on day 42 or day 56 as indicated in Table I. The chickens were weighed regularly t’hroughout the experiments. Clinical signs and mortality were recorded during the acute phase of each infection and total oocyst counts were made during the patent period of the disease.
370
HEIN
TABLE l’he Average
III
l’ofal Daily Oocyt Production per Bird, Calculated to the Nearest Million Ooc!/sts, Reinfecfion wi/h, Eimeria maxima or Cross Tnfeclion with Xmerin hrlmetti Susceptible chickens
Experiments
Group : species: dose :
2 E. maxima
4 R. brunetti
E. maxima-immunized
I-
3 I? R. maxima brunetti
After
chickens
5 E. brwzetti
7 R. , brunetti
-
J20,OOO1 51DO,00 1,250 __ -~
50,000
J20,OOO 510,000
,-a
l)ny
5 0 7 8 9 10 Total 5 0 7 8 9 10 Tot al
3.3 18.0 1.7 0.1 + 23
8.0 14.2 2.0 0.4 0.1 25
14.2 11.2 3.5 0.4 + 29
0.4 17.3 4.7 0.4 0.2 23
22.0 27.0 3.5 1.1 0.3 54
+ 13.2 7 (i 1.8 0.3 0.1 23
Ilay
l)ay
20,000
-
16.8 13.8 2.7 0.8 + 34
~
14.0 7.1 1.8 0.2 + 23
+ 14.4 9.4 2 (i 1.1 0.2 28
18.5 5.0 1.8 0.4 0.1 26
-
5
10 Totztl
2ti. 0 9.0 3.0 1.2 i 0.1 ~ 39
19.0 5.7 2.9 0.2 + 28
0.5 16.0 4.0 0.2 0.2 21
(i 7 8 9
a + oocysts
detected
on salt flotittion,
number
too small to comet.
RESULTS
Effects of the immunizing infections. After the first infection no sign of anorexia or depression was noted although growth was slightly retarded between day 5 and day 7 (Fig. 1) and oocysts were produced during the patent phase of the infection (Table II) . After the second and third infections no detrimental effects on growth were detected, although the mean weight of the immunized chickens was still, but not significantly, less than that of the noninfected chickens. Oocysts were not detected in the feces of
the chickens after the second infection. A few oocysts were present in the feces of the chickens given the highest dose in the third infection, between day 34 and day 37 in Experiment 3 (Table II). Effects of the challenge infections with E. maxima. After reinfection with the high challenge doses of oocysts on day 42 (Experiments 1 and 3) or day 56 (Experiment 2) no evidence of infection was detected in the immunized chickens in group 3. Typical signs of coccidiosis, however, were seen in the previously uninfected chickens in group 2 (Tables III and IV, Figs. 2 and 3). Effects of the cross infection with E. bru-
E. brunetti TABLE
371
I\’
R. nza.~ime-immunized chickens
Susceptible chickens Esperiment
Group : species: dose:
R.
brunetti ~0,000
1
2
Clinical + signs Mortality Cl0Weight lossc oocyst x ; 10” ~ Clinical signs I 1lortalitJ 67 ,o Weight loss oocq-sts 106
1,250 ++++I
+‘I
I,‘10 13
~ 3 23
I-t+++’
++
++++ ‘.‘VI0 13
1 ‘10 16 23
Ii -+++
/++++I ~
2 10 ‘1
1
a ++ marked anorexia atld depression. b ++++ complete anorexia, very high morbidity. c The mttxim~m~ difference it1 weight betwren the immunized or slwceptible chickells atid the llonix,fected chickens on the 6th or 7th day after challenge or cross infection expressed as a percentage of the weight of the rloninfected chickells.
netti. In all three experiments typical signs of acuic coccidiosis occurred in both the susceptible control chickens (groups 4 and 6) and the chickens immunized to E. maxima (groups 5 and, 7) after infection with low or high doses of oocysts of E. brunetti. There was no significant difference between the severity of infection in the susceptible control chickens and t.he immunized ehickens given similar doses of oocysts of E. brunetti. The pathogenic effects of E. brunetti appeared similar in chickens immunized ei-
ther in an identical manner t’o that used by Rose in 1967 (Experiment 3) or with doses of oocysts of E. maxima as used at Weybridge (Experiments 1 and 2). Morbidity was high, deaths ranged from 10 to 40% and characteristic lesions of hemorrhagic enteritis were found in birds which died. On day 5 and 6 cyanosis was marked, anorexia was complete, and the birds tended to sit on the pen floor with quivering tail feathers: dysentery was severe. The mean weight fell on the 5th, 6th, and 7th days after infection
372
HEIN
KEY DOSE OF OOCYSTS x16 42
42
NIL
NIL
NIL I
NIL
.-a NIL NIL O-0 1.25 5
NIL 20
320 320
c-a ‘NIL
c-2
E.BRUNETTI
E.MAXIMA 14 28
DAY0
NIL NIL
x-x
1.25 5
M
125
x-*
1.25 5
20
20
0
NIL NIL w. NIL
1.25
Y .-.
+2
20
10
12
FIG. 2. The mean weight of the chickens after challenge with oocysts of E. mtima cross infection with oocysts of E. brunetti (Experiment 1).
and was still lower than the noninfected chickens on the 12th day (Figs. 2 and 3). After adjustment by covariance to allow for differences in mean weight on the 3rd day, the mean weights of the infected chickens
or
were significantly lower than the noninfected birds on the 7th day (P = 0.001) and the 12th day (P = 0.01). Small differences in weight were apparent between certain groups after infection with E. brunetti
373
E. brunetti KEY
but they were not significant. Total oocyst production varied slightly between groups receiving the lowest dose of 1,250 oocysts of E. brmetti but there was little difference in oocyst output of chickens receiving the higher challenge doses of 20,000 and 50,000 oocysts of E. brunetti (Table III).
DOSEOFOCCYST5XlO’
I4
28
42
42 NIL
w
NIL
NIL
NIL
NIL
. .
NIL
NIL
NIL
50
ao5 AA
50xX) NIL
x-x 5
DISCCSSION The results showed conclusively that chickens immunized with oocysts of E. maxima were fully prot’ected against a high challenge infection wit’h the same species but that they were completely susceptible to cross infection with low or high doses of oocysts of E. brunetti. The results were similar in chickens immunized, challenged, and cross infected with different numbers of oocysts. The results of the present study using the Houghton strains of E. brunetti and E. maxima were identical with those of earlier work using the Weybridge strains of these species. There was no evidence of cross immunity (Tables III and IV) and these findings therefore support the observations of Tyzzer, Theiler, and Jones (1932) who considered that resistance to reinfection with Eimeria sp. was species specific. These results directly disagree with the findings of Rose (1967). Analysis of the data reported by Rose (1967) shows that large numbers of oocysts were passed after both the first and the second immunizing infections. This result differs significantly from that of Rose and Long (1962) in which oocyst production was negligible after the second immunizing infection with E. maxima and from current immunological studies at Weybridge employing multiple infections in which oocyst production was also minimal after the second and third immunizing infections with either E. acervulina, E. brunetti, E. maxim.a, or E. necatrix. The discrepancies connot be due to strain variation between the Houghton and Weybridge strains of E. maxima and E. brunetti, nor is it likely
E.BRUNETTI
EMAXIMA
MY0
NIL
50 50
NH
50
50 KM
p”
c-2
0
+2
4 au5
6
rp
8
10
FIG. 3. The mean weight of chickens after challenge with oocysts of E. maxima or cross infection with oocysts of E. brunetti (Experiment 3).
that differences in experimental diet or breed of bird could be responsible for such a marked difference in oocyst production. Howercr, the high product’ion of oocysts
374
HEIN
after the second immunizing infection in Rose’s experiment could be an indication of an adventitious infection at this time, which, if it were E. brunetti, would explain the apparent immunological relationship between E. brunetti and E. maxima and the complete failure in the present study using the Houghton strains of Eimeria to confirm the work of Rose (1967). ACKKOWLEDGMENTS Acknowledgments are due to Dr. M. E. Rose who kindly supplied suspensions of oocysts of the Houghton strains of E. brunetti and E. mnximn. The author thanks Mr. S. F. M. Davies and Dr. L. P. Joyner for their helpful discussions during this study and Miss N. Hebert for her statistical analysis of the results. I acknowledge the cooperation of Mr. R. Sayer in the preparation of Figs.
l-3 and thank Mr. C. Norton and Mr. P. Hall for their invaluable technical assistance. REFERENCES HEIK, H. E. 1968. The pathogenic effects of Eimeria aceruuliw in young chicks. Experimental Parasitology 22, l-11. JOYS-ER,L. P., .~NDDAVIES. S. F. M. 1960. Detection and assessment of sublethal infections of Eimeria tenella and Eimeria necatrix. Experimental Parasitology 9,243-249. ROSE, M. E. 1967. Immunity to Eimeria hnetti and Eimeria maxima infections in the fowl. Parasitology 57,363-370. ROSE, M. E., AND LOKG. P. L. 1962. Immunity to four species of Eimeria in fo\&. Immunology 5,79-92. TYZZER,E. E.. THEILER, H., ASD Jo~ves.E. E. 1932. Coccidiosis of gallinaceous birds. II. A comparative study of species of Eimerin of tht: chicken. American Journal of Hygiene 15,319393.