EXPERIMENTAL
42, 129-141
PARASITOLOGY
(1977)
Eimeria tenella: Skin Antigen
Hypersensitivity in the Fowl
to Injected
M. ELAINE ROSE Houghton
Poultry Research Station, Houghton, PEl?
(Accepted ROSE,
M. E. 1977. Eimeria
Experimental
Parasitology
2DA,
England,
for publication
Huntingdon,
Cambs.
U.K.
9 November
1976)
tenek: Skin hypersensitivity to injected antigen in the fowl. 42, 129-141. The skin hypersensitivity of fowls injected in the wattle with different antigens prepared from the protozoan parasite Eimeriu tenella was measured at intervals throughout immunization by infection and also after the injection of antigens in Freund’s complete adjuvant (FCA). In the early stages of immunization by infection, i.e., from 3 to 8 weeks after the initial inoculum of oocysts, the maximum response was found 4 hr after injection of antigen and was probably Arthus in type. At its peak, this response coincided with the presence of precipitating serum antibodies and was transferable with serum. A smaller delayed response was als’o present and, in the later period of testing (from approximately 10 to 18 weeks), ‘this became much more marked whereas the 4-hr response had declined and was negligible or absent. Thus, with the method of immunization used here, once immunity became complete (i.e., no production of oocysts after a challenege inoculum), there was a change from predominantly a 4-hr response to a delayed type. However, a substantial degree of immunity was present in infected birds which had strong 4-hr responses but weak 48-hr responses. A course of injections of antigens (prepared from the parasite) in FCA induced wattle hypersensitivity but no reduction of oocyst production after a challenge inoculum. INDEX DESCRIPTORS: Eimeria tenella; Coccidiosis; Protozoan parasite; Hypersensitivity; Skin testing; Immunity; Chicken; Antigens; Delayed type hypersensitivity; Immunization; Arthus response.
in coccidian infections. Positive macrophage migration inhibition tests (MMIT) Immune responses to infections with cochave been obtained with splenic (Morita, cidia, as with many other pathogens, are Tsutsumi, and Soekawa 1973) and pericomplex. Those mediated by antibodies toneal exudate (Rose 1973) ceils in the have been fairly extensively investigated presence of specific antigen, and a role for and there is evidence that they are intransfer factor (TF) has been described in volved, at least to some extent, in protecEimeria nieschulzi infections in rats (Lition. However, often, especially when comburd, Pabst, and Armstrong 1972) and in plete immunity has been established, no antibody activity may be demonstrable, E. bovis infections in cattle (Klesius, and it is possible that at this stage immune Kramer, Burger, and Malley 1975). The responsesof the cell-mediated (CMI) type demonstration of delayed hypersensitivity may assume a greater role, but there is (DH) responses, long considered to be little direct evidence for CM1 responses synonymous with CMI, to the parasite anINTRODUCTION
Copyright All rights
0 1977 by Academic of reproduction in any
I’rcss, Inc. form reserved.
ISSN
0014~ 4894
130
M. ELAINE
tigens would provide further evidence for CM1 responses but, until recently, there have been no published ,descriptions of hypersensitivity reactions in the skin. Delayed hypersensitivity to coccidian oocysts has now been demonstrated in rabbits infected with E. stiedui (Klesius, Kramer, Frandsen 1976) and in calves infected with E. bovis (Klesius, Kramer, Burger, and Malley 1975). Reference to unpublished work by Rose in a recent review (1973) indicated some success with fowls infected with E. tenella or E. maxima, but in the initial stages of immunization reactions of a more immediate type were commonly obtained. As there is evidence that in fowls the type of immune response to’ coccidia changes during immunization, an examination ‘of skin hypersensitivity throughout infection and immunization was attempted. The results of experiments in fowls infected with E. tenella and skin tested with various antigens prepared from the parasite are described below. MATERIALS
AND METHODS
Animals Unsexed chickens of the HPRS Light Sussex strain maintained coccidia free until experimentally infected at 2-4 weeks of age were used throughout. They were fed a diet formulated by the Coccidiosis Discussion Group (see Ryley and Betts 1973). Parasite This was the Houghton (H) or embryoadapted (TA) (Long 1972), nonpathogenie strain of Eimeria tenella. Infection and Consequent Immunization with E. tenellu: Expts. l-7 Groups of chicks (2 to 4 weeks old) were given a primary infection with 50,000 or 100,000 oocysts, orally, of the nonpathogenie strain of E. tenda (TA) faollowed,
ROSE
at 2-week intervals, by two (Expts. 2 and 5) or three (all other Expts.) inocula of SO;000 oocysts of E. tenella (H) to induce complete immunity, i.e., no production of oocysts after a challenge inoculum. Antigens (i) For testing. The majority of the tests were made with antigens prepared from suspensionsof E. teneZZa (H) oocysts (00 antigen). Thoroughly washed sporulated oocysts, after salt flotation, were treated with 10% hypochlorite solution at room temperature for 5 min to remove any adherent bacterial, fecal, or other ‘debris together with the outer layer of the oocyst wall ( Wagenbach, Challey, and Burns 1966). After thorough washing, they were suspended in phosphate buffered saline (PBS ), pH 7.0, to a concentration of 10 X 106/ml and agitated with glass beads ( appro’x. 500 pm in .diameter) on a “Whirlimixer” ( F ison’s Scientific Apparatus, Loughborough, Leicestershire, England) until thoroughly broken down. The material was pipetted off the beads and then frozen and thawed repeatedly to ensure complete disruption, The supernatant solution (optical density approximately lo-15 units/ml at 280 pm) removed after centrifugation (twice for 10 min at 5000g room temperature) was stored in l- t’o 2ml aliquots at -20 C until required for use. A limited number of tests was made with antigens prepared from merozoites ( Mz antigen). Merozoites were obtained from embryos killed 5 days ‘after inoculation with 80,000 sporozoites of E. tenella (H). Heavily infected areas in the chorioallantoic membrane were cutout and incubated in 0.25% trypsin bile solution at 40 C for 15 min. The merozoites were recovered after filtration through stainless steel mesh (approx. aperture 100 pm), washed in PBS, and made up to a final concentration of 38 x 10s/ml before storing at -20 C. Allantoic fluid (IAF antigen) from 15-day embryos was harvested
Eimeria
ten&a:
HYPERSENSITIVITY
96 hr after inoculation with approx. 20,000 sporozoites of E. tenella (H), centrifuged (as above), and the supernatant soIution was stored at -20 C until required for use. (ii) For injection in FCA: Expts. 8-10. (a) A mixture of 00 and IAF antigens (3 ml/bird) in the ratio of 1:2 (Expt. 8) or 1: 1 (Expt. 9) was emulsified with an equal volume of Freund’s complete adjuvant (FCA) and injected intraperitoneally (ip) once into 4-week-old birds. (b) Mz antigen (1 ml/bird) was emulsified with an equal volume of FCA and injected ip three times at intervals of 2 weeks (Expt. 10). The first injection was given when the birds were 2 weeks old. lntradermal
Tests
IN THE FOWL
131
gens in FCA were injected intraperitoneally, some birds were tested twice (see below). For histoological examination wattles were fixed in formal saline and stained with hematoxylin and eosin. Tests In Vitro Macrophage migration inhibition (MMI) tests were carried out as previously described (Rose and Hesketh 1974) and sera were tested for precipitating antibodies by diffusion in agar gel (Ouchterlony method) containing 8% NaCI. Preparation. of Cell Suspensions Spleens and cecal tonsils were removed aseptically and placed in medium 199 containing penicillin, 40,000 units/100 ml, and streptomycin, 60 mg/lOO ml. After teasing with scaIpe1 bIades in fresh medium and filtration through stainless steel mesh (approx. aperture 100 pm) the numbers of cells were estimated in a hemocytometer and their viability was determined by the exclusion of trypan blue.
These were done, as is customary for tuberculin testing in the fowl, in the wattle. Antigens in 0.05-1~1 aliquots were injected intradermally into the wattle on the left hand side (LHS ) of the animal, and the same amount of PBS was injected on the right hand side (RHS) using all-glass tuberculin-type syringes and 27-gauge needles, In young birds with undeveloped wattles, injection and measurement are Design of Experinwnts difficult, and in older birds injections of (i) Birds immunized by infection. In the very thin but tough wattle dermis are not always truly intradermal (see Bush- Expts. l-4 three or more infected birds nell and Brandly 1931) but can be checked were tested at each time interval (usually of 2 weeks) from l-11 weeks after the iniby the appearance of blanching and a firm tial inoculation, by which time all the bleb. The hyperemic nature of the wattle also causes difficulties and precludes ob- birds had been used. In Expt. 5, birds servations other than measurements of were tested from Week 1 through to Week thickness. Readings were taken 20 min and 18 after inoculation, and in Expts. 5, 6, and 7, in order to obtain more ,data on at 4, 24, and 48 hr after .antigen injection. later responses,birds were tested at weekly Wattle thickness was measured with ordinary calipers (Expts. l-5) or, in the case intervaIs from Week 11 through to Week 18. Untreated coccidia-free controls were of the older birds, (Expts. 6 and 7) with dial g&uge calipers (“Pocotest A02,” Ca- also tested at each time interval. (ii) Birds injected with antigen in FCA: robronze Ltd., School Road, London W4). The r&&s, given in millimeters, represent Expts. 8-10. Birds were wattle-tested 2 the difference in thickness between the weeks after the last injection ‘of antigen and after the 4%hr reading, they were RHS and LHS wattles. In most experiments (l-7), birds were skin-tested once inoculated with oocysts orally and again only, but in experiments (S-10) where anti- wattle-tested 2 weeks later.
1: 1)
1:2)
0
26
30
0 0
9
0
10
10
Number of birds tested
tenella Oocyst
5 Tested approximately 2 weeks after last injection b Tested 2 weeks after oral inoculum. c Approximately 250 oocysts of H strain or 100,000 d Not done.
None
+ FCA
Saline
+ FCA
(Me)
Antigen
10
+ FCA
Saline
+ IAF,
injected
to Eimeriu
Antigen (00 + IAF, +FCA Saline + FCA None
(00
Antigen +FCA
Material
Responses
9
8
Experiment number
Wattle
oocysts
course
after
00
00
of TA
+ IAF)
of antigen.
(00
Test antigen
After
Antigen
TABLE
strain
I
given
0.1
0.2
0.1
orally
0
0.7
0.5
ND
2 weeks
0.2
0.4
0.4
0
after
and after After
Challenge
00
00
00 Mz IAF 00 MB IAF 00 Ms IAF
Test antigen
challenge
Infection
of antigen.
12 12 11 11 11 12
10 10
10
Number of birds tested
last injection
H TA H TA H TA
H
H
Oocyst inoculum strainc
in FCA
0.5
48
4
0.3 NDd
test
of reading (hr)
Time
in wattle (mm)
of Antigens
Difference thickness
of injection9
ip Injection
8-10)
0.5 0.7 0.6 0.5 0.1 0.2
0.1 0.1
0.8
ND
0.3
Time
1.2 1.0 1.4 0.9 0.3 0.3
0.5 0.5
1.0
ND
4
0.6 0.5 0.6 0.3 0.3 0.3
ND
0.1
0.6 0.4 0.2 1.1 0.2 0.1 1.0 0.5
48
test
in wattle (mm)
of reading @r)
Difference thickness
infectionb
(Expts.
g “M
I3
R M g
Eirneria
ten&a:
HWERSENSITIVITY 133 THE FOWL
RESULTS Immunization by Infection Eimeria tenella
with
The initial inoculum of E. tenella (TA) protected well against the pathogenic effects of E. tenella (H) in all experiments except Expt. 1 in which there was some blo’od in the feces after the first inoculum of E. ten&a (H). In Expts. 3-6 no oocysts were detected in the feces after the final inoculum of E. tenalla (H) (given at Week 4 ‘or 6) indicating that the birds had become completely immune; small numbers were detectable by salt flotation in Expts. 1 and 2. Immunity was again tested (Expt. 6 only) on Week 18 when four birds were given 50,000 moocysts of E. tenella (H); they did not show any clinical signs of disease but some soocysts were found in feces examined by salt flotation. Four control susceptible birds given the same inoculum suffered severe infection, with blood in the feces on Days 5 and 6 after oocyst inoculation, and large numbers of oocysts on Days 7 and 8. Attempted
Immunization
by Injection
of Antigens in FCA
The results of challenge with oocysts of E. tenella (H) or ( TA ) (to be given in detail elsewhere) were similar in all groups, whether injected with antigen in FCA, saline and FCA, or untreated. Thus no protection was afforded by the administration of these antigens. Skin Hypersensitivity ii) With Diferent
Antigens
Of the antigens tested, oocyst (00) produced in similarly treated birds the greatest responses and caused minimum reactions in coccidia-free animals. As this antigen was also the most easily obtainable it was used in the majority of the tests. Reactions to oocyst, merozoite, and IAF antigens in the same group of infected
133
(but other wise untreated) birds may be compared in Table I (Expt. 8, third group). In most cases delayed responses at 24 and 48 hr were similar, with a tendency to be greater at 48 hr. (ii) During and After Immunization by Infection Oocyst antigen was used throughout, but in different preparations in some of the experiments. The results for Expts. 1-4 (observations from Weeks 1 to 11) were pooled, as were those for Expt. 5, 6, and 7 (observations from Weeks 11 to 18); data for Expt. 5 are given separately (Weeks l18). Readings obtained from untreated control birds in all experiments were pooled (Fig. 1). Mean values obtained from readings at 20 min were low ( < 0.3 mm) and variable, but there were little or no differences between infected and control groups, especially from 11 to 18 weeks. Readings obtained at 24 and 48 hr were somewhat similar, hence the data for 24 hr are omitted from Fig. 1 in which the results obtained at 4 and 48 hr are given, together with the numbers of birds tested at each time interval. Reactions at 4 hr. A distinct pattern of response was obtained at this time interval. Values increased sharply to a peak at 4 to 7 weeks and then declined, again quite steeply, to low values at 11 weeks and thereafter. The highest mean values obtained represented ‘approximately a 100% increase in wattle thickness, but in a few individuals increases of between 150 and 200% were found, Reactions at 48 hr. Positive responses were obtained from the second week after infection, remaining at a fairly low to moderate value until Week 9 after which they increased quite considerably. From 11 to 18 weeks the values remained high but somewhat variable, while those obtained in the same birds at 20 min or 4 hr were particularly low. Maximum mean
134
M.
d 5
ELAINE
ROSE
1.0 -
.E
0.9
-
e 5 8 g
@7-
At 48hrs
3
0.5 -
0
Y 1
I
I
Y
3 Weeks
I 5
I
after
Y 7 initial
9
11
13
15
17
inoculum
FIG. 1. Dermal reactions 4 and 48 hr after injection of Einzeria tenella oocyst antigen in birds immunized by infection and in coccidia-free controls. Mean values are given, in millimeters, for differences in thickness between right hand (injected with antigen) and left hand (injected with saline) wattles. Numbers of birds tested at each time interval are given beside symbols. Immunized birds: pooled results of Expts. l-4, c]- - -0; results of Expt. 5, A. coccidia-free controls : pooled results, O-O; pooled results of Expts. 6 and 7, A----
o- -0. values represented again approximately a 100% increase in wattle thickness. The change in character Iof the response with time after the initial infection may be seen in the results (given in Fig. 2) for birds of Expt. 5 tested at Weeks 5 and 11. The histol.ogical features of the responses at 4 and 48 hr are shown in Fig. 3. The 4-hr reaction is one of acute inflammation
with edem.a and an infiltration of heterophi1 polymorphonuclear leucocytes. At 48 hr the reactions were characterized by an intense infiltration with small mononuclear cells, (iii) In Chickens Infected with Antigens in Freund’s Complete Ad/uvant Birds were wattle-tested approximately 2 weeks after the last antigen injection,
Eimeriu
&%?kz‘a:
HYPERSENSITIVITY
immediately preceding infection by oral inoculation with oocysts, ,and again 2 weeks later. The results are given in Table I. At the first test, wattle reactions were positive in all groups injected with antigen in FCA; 20-min values were negligible or low and the highest readings were obtained at 4 hr, but some delayed response was probably also present. Control groups injected with FCA and saline gave negative ,or negligible values although there was some indication of a possible delayed response at 48 hr in this group in Expt. 10. A challenge infection given to antigeninjected groups appeared to enhance the responses, particularly at 20 min, and in Expt. 10 groups previously injected with FCA and saline gave higher values than untreated groups. However, it is possible that these results were due to sensitization of the wattle by the previous testing. Transfer
of Skin Hypersensitivity
Limited attempts skin reactivity with (i) With serum.
were made to transfer serum and with cells. Two pools of serum
IN
THE
FOWL
were used for transfer experiments. One was ‘obtained from the birds of Expt. 4 bled 5 weeks after the initial inoculum of oocysts when the response at 4 hr was higher th,an at 48 hr, and the other was from three birds of Expt. 7 whose 4%hr responses were high and 4-hr responses very low (bled 14 weeks after the initial inoculum of oocysts ) . Five milliliters of the pooled serum were injected intraven’ously into each of three or five 3-week-old chickens, and 1 hr later antigen or saline was injected into the wattles. A control group of two birds was similarly treated after receiving serum from coccidia-free chickens. The results of wattle measurements made at 4, 24 and 48 hr later are given in Table II, together with the responses of the serum donors tested immediately before they were bled. In two of the three birds given the “early” serum, a low positive result was obtained at 4 hr; readings at 24 or 48 hr gave negligible or negative values. Thus, although only small numbers of animals were used, the 4 hr response did seem to be transferable with serum.
Week 11
Time. hours
after
135
injection
of wattle
FIG. 2. Values, mean and range, for dermal reactions (differences in thickness between wattle injected with antigen and wattle injected with saline) at different times after injection of Eimeria tenella oocyst antigen. Results from Expt. 5; tests carried out at 5 weeks (three birds) and at 11 weeks (three birds) after the initial inoculum of oocysts. Infected birds: n-----n; coccidia-free controls (three birds) : O- - -0.
136
M.
ELAINE
ROSE
&‘W??‘iU
tiXl&X
HYPERSENSITIVITY
Skin reactivity was absent at all test interin the birds which received the “late” serum (high delayed responses in donors) and in those which were given control serum. (ii) With cells. Suspensions of cecal tonsil and spleen cells were prepared from three birds killed 11 weeks after the initial inoculum of oocysts when the wattle responses were of the delayed type (mean difference in wattle thickness = 0.1, 0.6, ‘and 1.0 mm at 4, 24, and 48 hr, respectively) and from three control, coccidiafree birds (wattle responses negative). Each suspension (approx. 50 x lOa spleen cells or 2 X lo8 cecal tonsil cells) was injected intraperitoneally into a single 3week-old recipient and tests were done 4.25 hr later. No differences in thickness were apparent when the wattles were measured at 4, 24, and 48 hr; the responses seen in the donors had not been transferred to the recipients. vals
Correlation of Skin Hypersensitivity In Vitro Tests
with
(i) After immunization by infection. Pooled serum samples were obtained at weekly intervals from Expts. 2, 4, and 5 and tested for precipitating antibodies wth ‘oocyst antigen. Precipitin bands were found only with serum samples ‘obtained at Weeks 3, 4 ( Expt. 2, 4, and 5), and 5 (Expts. 2 and 4); all other samples gave negative results. Thus the presence of precipitating serum antibody coincided with the period of rapid increase and peak in the 4-hr response, but not with the decline phase. Tests for inhibition of macrophage migration in vitro in the presence of antigen were carried out on some ,of the animals whose wattle responses are summarized in
FIG. 3. Sections of wattles after injection of Eimeria weeks after initial inoculum eosin.
IN
THE
Fig. 1. In most cases peritoneal exudate (PE) cells were obtained from the birds immediately after the 48-hr wattle readings were taken, the antigens used being oocyst and merozoite. The results of these tests were highly variable; a small number of positive tests was observed, but the results did not correlate with wattle sensitivity. (ii) After injection with antigens in FCA. Serum samples were taken from the birds in all three experiments of this type after the course of antigen injections (immediately before oocyst inoculation) and again 2 weeks after ‘oocyst inoculation, and tested f.or the presence of precipitating antibodies with various antigens. The findings, given in Table III, are in fair agreement with the 4-hr wattle responses (Table I) in that (a) positive results were obtained in ‘antigen injected groups; these were enh,anced after infection and considerably stronger than those found in other groups, and (b) in Expt. 10 the group injected with FCA and saline which after infection gave higher wattle responses than the untreated groups, also had a greater proportion of birds with precipitating antibodies. Surprisingly, some sera of this group gave positive reactions before infection although there was no fecal evidence of extraneous infection. A few birds were used for MM1 tests after the output of oocysts from the infection with E. tenella (H) had ceased; results are given in Table III. In Expt. 10, leucocytes were used for some birds because of the difficulties ‘of obtaining large enough numbers of PE cells in older birds, especially after a series of intraperitoneal injections with FCA. A degree of inhibition was seen in a small number of infected (‘overall 5/15) birds, but there did
of control (A-D) and infected (E-H) chickens antigen or saline. Wattles injected 4 (E, of oocysts. Fixed in formal saline, stained with
tenelk
137
FOWL
taken 4 or 48 hr F) or 9 (G, H) hematoxylin and
138
M.
Transfer
of 4-hr
Skin
Serum Time of bleeding (weeks after initial oocyst inoculum)
ROSE
TABLE
II
to Eimeria
tenella
Oocyst
donors
Difference Time
Skin reactivity : in wattle thickness (mm) of reading test (hr) 24
48
5
0.8
0.4
0.5b
14
0.1
0.9
0.9c
0
Antigen
with
“Early”
Serum
Recipients”
4
0 Control coccidiafree a Each b Mean c Mean
Reactivity
ELAINE
0.1
0
Bird number
Difference Time
Skin reactivity: in wattle thickness (mm) of reading test (hr)
4
24
48
1 2 3 1 2 3 4 5 1
0.3 0.2 0 0 0 0 0 0.1 0
0.1 0.1 0 0 0 0.1 0 0 0
0.1 0 0
2
0
0
0
0 0 0
3-week-old bird given 5 ml of serum intravenously; antigen or saline injected 1 hr later. values from three birds tested at random from group of 20 which provided the serum pool. values from the three birds which provided the serum pool.
not seem to be any marked consistent differences between the groups. However, the numbers examined were small. DISCUSSION
The results of the tests for skin reactivity are expressed only in terms of wattle thickness, no other observations being possible because of the hyperemic nature of the wattIe. In highly reactive wattIes some temperature .difference may be apparent but this w.as not detected in the system used here. Hypersensitivity After Infection
Reactions
During
and
In the five experiments in which birds were tested at intervals during 11 weeks after an initial inoculum of Eimeria tenella (TA) followed by two or three inocula of E. tenella (H), the 4-hr reaction was the most intense and showed a ,definite rise and fall during the period of test. The pattern was similar to that previously found for serum cytophilic/opsonic antibody ac-
tivity (Rose 1974). Peak responses were noted at a time (Weeks 4-7) when protective activity has been demonstrated in serum (Rose and Long 1971) and, in experiments 2, 4, and 5, precipitating antibodies were found in the pooled serum samples at the time of the peak and in the 2 weeks before. In addition, 4-hr reactions appeared to be transferable with serum taken at Week 5. Thus this response was probably of the Arthus type. In the latter half of the test period the 4-hr response was negligible. Delayed reactions were also present from the second week after the first oocyst inoculum remaining at a moderate, fairly constant level until Week 9, after which they became much more marked. This increase coincided with the virtual disappearance of the early reactions. Unsuccessful attempts to transfer the delayed response with suspensions of spleen and cecal cells at Week 11 were probably due to histoincompatibility between donors and recipients, as was a previously reported failure to transfer delayed hypersensitivity
Eimeria
tenella:
HYPERSENSITIVITY
1976). A mild Arthus-type response was also present and this was transferable by serum, whereas the ,delayed response was transferred only with mesenteric lymph node cells. Calves infected with E. boois gave delayed reactions at 26 days after infection (Klesius, Kramer, Burger, and Malley 1975). The earlier development of predominantly delayed responses with these tw,o species, compared with E. teneZla in fowl, may be related to differences in immunogenicity. E. tenella is, with E. necatrix, one of the least immunogenic of the species parasitizing the chicken (Rose and Long 1962) and probably very poor in this respect in comparison with species parasitizing other hosts. The type of antigen used may also be a factor in determining the nature of the response obtained; in the work described here only soluble antigens prepared by physical means were used, whereas Klesius, Kramer, Burger, and Malley (1975) and Klesius, Kramer, and Frandsen (1976) used a particulate antigen prepared from unsporulated oocysts by TCA precipitation
reactions which otherwise fulfilled all the criteria applied to this form of immune response (see Warner, Ovary, and Kantor 1971). Histologically the reactions were typical of the delayed type and they were not transferred with serum (Table II). The results of MM1 tests did not provide any in vitro evidence of a CM1 response during this latter half of the test period, but the numbers examined were small, and it is possible that, as with leishmaniasis and toxoplasmosis in guinea pigs, positive results may be obtained only when the delayed skin reaction is very high (Tremonti and Walton 1970). Alternatively it is possible that as the cells for MM1 tests were obtained at the height of the wattle response (48 hr after injection of antigen), all the sensitized cells were mobilized to the wattle and therefore not available in the PE (see Granlund and Loan 1974). Responses of the 20-min type were low throughout the period of testing. In rabbits infected with E. s&due delayed reactions appeared to predominate earlier (Klesius, Kramer, and Frandsen TABLE Results of Tests for Precipitating Migration in Groups Injected Material
Experimerit number
injected
After
III
Serum Antibodies and for Inhibition with Eimeriu tenella Antigens in FCA course
139
IN THE FOWL
of injections
Precipitin test? Number positive with
dfter 00cgst strainb
of Macrophage (Expts. 8-10) challenge
infection
Precipitin test Number positive
:a with
test
MMITc antigens
-
8
Antigen (00 + IAF, + FCA Saline -I- FCA
IAF
Ma
00
O/10 o/10
IO/10 O/IO
10/10 O/IO
10
None, not infected Antigen (00 + IAF, 1: 1) +FCA Saline + FCA None None, not infected Antigen (Ma) + FCA Saline
+
FCA
None None,
not infected
MB
00
s/10 o/10 Q/Q
lO/lO o/10 Q/O
lO/lO 3/10 3/10
ND
lO/lO 4/10 3/10
Mz
00
l/2
o/2
O/l O/l O/l
l/l l/l O/l
1:2) H
None 9
IAF
X/IO
H
ND
o/10
26/29
23/29
O/28
3/28
H TA H TA H TA -
ND
0 Birds bled 2 weeks after last injection of antigen or oral inoculation of oocysts, respectively. 6 Approximately 250 oooysts of H strain or 100,000 oocysts of TA strain given orally 2 weeks e Tested 3-5.5 weeks after oooyst inoculation. Migration index below 0.8 = positive.
12/12 12/12 9/11 9/11 2/11 6/12
l/2 l/2 O/l O/l Q/6
10/12 12/12 6/11 4/11 o/11 l/12
o/2 after
last
inj&ion
of antigen.
140
M.
ELAINE
followed by pepsin digestion and acid extraction. Different ‘methods of preparing antigens from Trypanosonuz brucei have been shown to give different results for 4-hr relative to 24-hr reactions (Tizard and Soltys 1971), and in T. cruzi-infected rabbits the injection of soluble or particulate antigen fractions induced immediate ‘or delayed type responses, respectively (Teixeira and Santos-Buch 1975). The importance of the type of test antigen has als’o been shown in a Toxoplasma gondii test ,system (Lai, Tizard, Quinn, and Ingram 1975). It is therefore highly likely that the choice of test antigen has an important bearing on the results obtained and these ought to be evaluated accordingly. However, in the work described here, in which an increase in delayed response was found later in immunization, the same test antigen was used through’out. A late increase in delayed-type skin hypersensitivity has been noted in infections with ,other protozoa. In humans infected with T. gondii, DH to toxoplasmin occurs late in infection (Frenkel 1948), although in guinea pigs it is found from 1 week after (Krahenbuhl, Blazkovec, and Lysenko 1971). In T. brucei-infected rabbits, there is a change over from a 4-hr Arthus type ,of response found at 2 weeks to a delayed 24-hr type found not earlier than 3 weeks after the onset of infection (Tizard and Soltys 1971) and in monkeys infected with Plasmodium knowlesi, delayed hypersensitivity was found in only one chronically infected animal which was tested considerably later than any of the others (Phillips, Wolstencroft, Brown, Brown, and Dumonde 1970). However, in Leishmania enrietti-infected guinea pigs, DH developed early (Bryceson, Bray, Wolstencroft, and Dumonde 1970). Hypersensitivity Reactions of Antigens in ECA
After
Infection
Wattles of birds which had received injections of antigens in FCA became hypersensitive to the immunizing and other
ROSE
coccidian antigens. The reactions were most intense at 4 hr but in three of the four experiments ‘there appeared to be also a delayed response as wattle thickness was maintained throughout the test period. After a challenge infection the responses were enhanced and slightly greater than in the oontrol group injected with FCA alone. There was also a tendency for groups injected with FCA alone to be more reactive than previously untreated groups. This may have been due to the effect of adjuvant, or possibly to previous testing of the wattle to determine the preinfection responses. On the other hand, in two experiments precipitin responses were also higher in this group. Precipitating antibodies were constantly found in the sera of antigen injected birds, but results .of tests for MM1 gave rather inconclusive results. From the evidence presented here, delayed hypersensitivity is present during the earlier stages of the acquisitJon of immunity but becomes more marked later on when immediate responses have diminished. Immunity ‘of the birds to reinfection was checked at Week 4 when the 4-hr response was high and the 48-hr response lower, and at Week I8 when the 4-hr response was absent and the 4%hr response high. A substantial degree of immunity was found at b’oth times and is thus associated with both types of response in animals immunized by infection. However, birds injected with antigens in FCA and showing reactions predominantly of the immediate type (together with high levels of precipitating antibodies) were susceptible to subsequent infection. Similarly, rabbits to which DH had been transferred by TF prepared from animals infected with Eimeria stiedae were susceptible to infection (Klesius, Kramer, Burger, and Malley 1975). Thus, although the skins of immune animals are hypersensitive to injections of antigens, dermal reactivity, immediate or delayed, is not necessarily an indication of immunity to infection.
i!?ime?iia
t&U&a:
HYPERSENSITIVITY
ACKNOWLEDGMENTS The excellent technical assistance of Patricia Hesketh and Barbara Fisher is gratefully acknowledged. Thanks are also due to my colleague, Dr. P. L. Long, for help with photography, and to Drs. P H. Klesius, T. T. Kramer, and J. C. Frandsen, USDA-ARS-SR, Regional Parasite Research Laboratory, Auburn, Alabama, U.S.A., for showing me their manuscript before publication.
REFERENCES BRYCESON, A. D. M., BRAY, R. S., WOLSTENCROFT, R. A., AND DUMONDE, D. C. 1970. Immunity in cutaneous leishmaniasis of the guinea pig. Clinical and Experimental Immunology 7, 301-341. BUSHNELL, L. F., AND BRANDLY, S. A. 1931. The reaction of the fowl to pullorum. Journal of the American Veterinary Medical Association 78, 64-78. FRENKEL, J. K. 1948. Dermal hypersensitivity to Toxoplasma antigens ( Toxoplasmins ) . Proceedings of the Society for Experimental Biology and Medicine 68, 634-639. GRANLUND, D. J., AND LOAN, R. W. 1974. Cellmediated immunity in the chicken: Cytotoxicity induced by specific soluble antigens. Journal of the Reticuloendothelial Society 15, 593-511. KLESIUS, P. H., KRAMER, T., BURGER, D., AND MALLEY, A. 1975. Passive transfer of coccidian oocyst antigen and diphtheria toxoid hypersensitivity in calves across species barriers. Transplantation Proceedings 7, 449-452. KLESIUS, P. H., KRAMER, T., AND FRANDSEN, J. C. 1976. Eimeria stiedai: Delayed hypersensitivity response in rabbit coccidiosis. Experimental Parasitology 39, 59-68. KRAHENBUKL, J. L., BLAZKOVEC, A. A., AND LYSENKO, M. G. 1971. In viva and in vitro studies of delayed-type hypersensitivity to Toxoplasma gondii in guinea pigs. Infection and Immunity 3, 260- 267. LAI, C. M., TIZARD, I. R., QUINN, P. J., AND INGRAM, D. G. 1975. An analysis of those components of Toxoplasma gondii responsible for delayed hypersensitivity reactions in mice. lmmunobgy 28, 611-620. Lmonn, E. M., PABST, H. F., AND ARMSTRONG, W. D. 1972. Transfer factor in rat coccidiosis. Cellular Immunology 5, 487-489. LONG, P. L. 1972. Eimeria tenella: Reproduction pathogenicity and immunogenicity of a strain maintained in chick embryos by serial passage. Journal of Comparative Patllology and Therapeutics 82, 429-437.
IN THE FOWL
141
MORLTA, C., TEXJTSU~I, Y., AND SOEI(AWA, M. 1973. Migration inhibition test of splenic cells of chickens infected with Eimeria tenella. Journal of Parasitology 59, 199-200. PHILLIPS, R. S., WOLSTENCROFT, R. A., BROWN, I. N., BROWN, K. N., .~ND DUMONDE, D. C. 1970. Immunity to malaria. III. Possible occurrence immunity to of a cell-meriated Plasmodium knowlesi in chronically infected and Freund’s complete adjuvant-sensitised monkeys. Experimental Parasitology 28, 339355. ROSE, M. E. 1973. Immune responses to the Eimeria: Recent observations. In “Proceedings of the Symposium on Coccidia and Related Organisms,” pp. 92-118. University of Guelph, Ontario, Canada. Rose, M. E. 1974. Immune responses in infections with coccidia: Macrophage activity. Infection and Immunity 10, 862-871. ROSE, M. E., AND HESKETH, P. 1974. Fowl peritoneal exudate cells: Collection and use for the macrophage migration inhibition test. Asian Pathology 3, 297-366. ROSE, M. E., AND LONG, I?. L. 1962. Immunity to four species of Eimeria in fowls. Immunology 5, 79-92. ROSE, M. E., AND LONG, P. L. 1971. Immunity to coccidiosis: Protective effects of transferred serum and ceils investigated in chick embryos infected with Eimeria tenella. Parasitology 63, 299-313. RYLEY, J. F., AND BETTS, M. J. 1973. Chemotherapy of chicken coccidiosis. Advances in Pharmacology and Chemotherapy 11, 221-293. TEIXEIRA, A. R. L., AND SANTOS-BUCH, CA. 1975. The immunology of experimental Chagas disease. II. Delayed hypersensitivity to Trypanosoma cruzi antigens. Immunology 28, 401-410. TEARD, I. R., AND SOLTYS, M. A. 1971. Cellmediated hypersensitivity in rabbits infected with Trypanosoma brucei and Trypanosoma rhodesiense. Infection and Immunity 4, 67P 677. Tremonti, L., AND WALTON, B. C. 1970. Blast transformation and migration-inhibition in toxoplasmosis and leishmaniasis. American Journal of Tropical Medicine and Hygiene 19, 49-56. WAGEIYBACH, G. E., CHALLEY, J. R., AND BURNS, W. C. 1966. A method for purifying coccidian oocysts employing clorox and sulfuric aciddichromate solution. Journal of Parasitology 52, 1222. WARNER, N. L., OVARY, Z., AND KAXTOR, F. S. 1971. Delayed hypersensitivity reactions in normal and bursectomized chickens. Znternational Archives of Allergy and Applied lmmunology 40, 719-728.
42, 129-141
PARASITOLOGY
(1977)
Eimeria tenella: Skin Antigen
Hypersensitivity in the Fowl
to Injected
M. ELAINE ROSE Houghton
Poultry Research Station, Houghton, PEl?
(Accepted ROSE,
M. E. 1977. Eimeria
Experimental
Parasitology
2DA,
England,
for publication
Huntingdon,
Cambs.
U.K.
9 November
1976)
tenek: Skin hypersensitivity to injected antigen in the fowl. 42, 129-141. The skin hypersensitivity of fowls injected in the wattle with different antigens prepared from the protozoan parasite Eimeriu tenella was measured at intervals throughout immunization by infection and also after the injection of antigens in Freund’s complete adjuvant (FCA). In the early stages of immunization by infection, i.e., from 3 to 8 weeks after the initial inoculum of oocysts, the maximum response was found 4 hr after injection of antigen and was probably Arthus in type. At its peak, this response coincided with the presence of precipitating serum antibodies and was transferable with serum. A smaller delayed response was als’o present and, in the later period of testing (from approximately 10 to 18 weeks), ‘this became much more marked whereas the 4-hr response had declined and was negligible or absent. Thus, with the method of immunization used here, once immunity became complete (i.e., no production of oocysts after a challenege inoculum), there was a change from predominantly a 4-hr response to a delayed type. However, a substantial degree of immunity was present in infected birds which had strong 4-hr responses but weak 48-hr responses. A course of injections of antigens (prepared from the parasite) in FCA induced wattle hypersensitivity but no reduction of oocyst production after a challenge inoculum. INDEX DESCRIPTORS: Eimeria tenella; Coccidiosis; Protozoan parasite; Hypersensitivity; Skin testing; Immunity; Chicken; Antigens; Delayed type hypersensitivity; Immunization; Arthus response.
in coccidian infections. Positive macrophage migration inhibition tests (MMIT) Immune responses to infections with cochave been obtained with splenic (Morita, cidia, as with many other pathogens, are Tsutsumi, and Soekawa 1973) and pericomplex. Those mediated by antibodies toneal exudate (Rose 1973) ceils in the have been fairly extensively investigated presence of specific antigen, and a role for and there is evidence that they are intransfer factor (TF) has been described in volved, at least to some extent, in protecEimeria nieschulzi infections in rats (Lition. However, often, especially when comburd, Pabst, and Armstrong 1972) and in plete immunity has been established, no antibody activity may be demonstrable, E. bovis infections in cattle (Klesius, and it is possible that at this stage immune Kramer, Burger, and Malley 1975). The responsesof the cell-mediated (CMI) type demonstration of delayed hypersensitivity may assume a greater role, but there is (DH) responses, long considered to be little direct evidence for CM1 responses synonymous with CMI, to the parasite anINTRODUCTION
Copyright All rights
0 1977 by Academic of reproduction in any
I’rcss, Inc. form reserved.
ISSN
0014~ 4894
130
M. ELAINE
tigens would provide further evidence for CM1 responses but, until recently, there have been no published ,descriptions of hypersensitivity reactions in the skin. Delayed hypersensitivity to coccidian oocysts has now been demonstrated in rabbits infected with E. stiedui (Klesius, Kramer, Frandsen 1976) and in calves infected with E. bovis (Klesius, Kramer, Burger, and Malley 1975). Reference to unpublished work by Rose in a recent review (1973) indicated some success with fowls infected with E. tenella or E. maxima, but in the initial stages of immunization reactions of a more immediate type were commonly obtained. As there is evidence that in fowls the type of immune response to’ coccidia changes during immunization, an examination ‘of skin hypersensitivity throughout infection and immunization was attempted. The results of experiments in fowls infected with E. tenella and skin tested with various antigens prepared from the parasite are described below. MATERIALS
AND METHODS
Animals Unsexed chickens of the HPRS Light Sussex strain maintained coccidia free until experimentally infected at 2-4 weeks of age were used throughout. They were fed a diet formulated by the Coccidiosis Discussion Group (see Ryley and Betts 1973). Parasite This was the Houghton (H) or embryoadapted (TA) (Long 1972), nonpathogenie strain of Eimeria tenella. Infection and Consequent Immunization with E. tenellu: Expts. l-7 Groups of chicks (2 to 4 weeks old) were given a primary infection with 50,000 or 100,000 oocysts, orally, of the nonpathogenie strain of E. tenda (TA) faollowed,
ROSE
at 2-week intervals, by two (Expts. 2 and 5) or three (all other Expts.) inocula of SO;000 oocysts of E. tenella (H) to induce complete immunity, i.e., no production of oocysts after a challenge inoculum. Antigens (i) For testing. The majority of the tests were made with antigens prepared from suspensionsof E. teneZZa (H) oocysts (00 antigen). Thoroughly washed sporulated oocysts, after salt flotation, were treated with 10% hypochlorite solution at room temperature for 5 min to remove any adherent bacterial, fecal, or other ‘debris together with the outer layer of the oocyst wall ( Wagenbach, Challey, and Burns 1966). After thorough washing, they were suspended in phosphate buffered saline (PBS ), pH 7.0, to a concentration of 10 X 106/ml and agitated with glass beads ( appro’x. 500 pm in .diameter) on a “Whirlimixer” ( F ison’s Scientific Apparatus, Loughborough, Leicestershire, England) until thoroughly broken down. The material was pipetted off the beads and then frozen and thawed repeatedly to ensure complete disruption, The supernatant solution (optical density approximately lo-15 units/ml at 280 pm) removed after centrifugation (twice for 10 min at 5000g room temperature) was stored in l- t’o 2ml aliquots at -20 C until required for use. A limited number of tests was made with antigens prepared from merozoites ( Mz antigen). Merozoites were obtained from embryos killed 5 days ‘after inoculation with 80,000 sporozoites of E. tenella (H). Heavily infected areas in the chorioallantoic membrane were cutout and incubated in 0.25% trypsin bile solution at 40 C for 15 min. The merozoites were recovered after filtration through stainless steel mesh (approx. aperture 100 pm), washed in PBS, and made up to a final concentration of 38 x 10s/ml before storing at -20 C. Allantoic fluid (IAF antigen) from 15-day embryos was harvested
Eimeria
ten&a:
HYPERSENSITIVITY
96 hr after inoculation with approx. 20,000 sporozoites of E. tenella (H), centrifuged (as above), and the supernatant soIution was stored at -20 C until required for use. (ii) For injection in FCA: Expts. 8-10. (a) A mixture of 00 and IAF antigens (3 ml/bird) in the ratio of 1:2 (Expt. 8) or 1: 1 (Expt. 9) was emulsified with an equal volume of Freund’s complete adjuvant (FCA) and injected intraperitoneally (ip) once into 4-week-old birds. (b) Mz antigen (1 ml/bird) was emulsified with an equal volume of FCA and injected ip three times at intervals of 2 weeks (Expt. 10). The first injection was given when the birds were 2 weeks old. lntradermal
Tests
IN THE FOWL
131
gens in FCA were injected intraperitoneally, some birds were tested twice (see below). For histoological examination wattles were fixed in formal saline and stained with hematoxylin and eosin. Tests In Vitro Macrophage migration inhibition (MMI) tests were carried out as previously described (Rose and Hesketh 1974) and sera were tested for precipitating antibodies by diffusion in agar gel (Ouchterlony method) containing 8% NaCI. Preparation. of Cell Suspensions Spleens and cecal tonsils were removed aseptically and placed in medium 199 containing penicillin, 40,000 units/100 ml, and streptomycin, 60 mg/lOO ml. After teasing with scaIpe1 bIades in fresh medium and filtration through stainless steel mesh (approx. aperture 100 pm) the numbers of cells were estimated in a hemocytometer and their viability was determined by the exclusion of trypan blue.
These were done, as is customary for tuberculin testing in the fowl, in the wattle. Antigens in 0.05-1~1 aliquots were injected intradermally into the wattle on the left hand side (LHS ) of the animal, and the same amount of PBS was injected on the right hand side (RHS) using all-glass tuberculin-type syringes and 27-gauge needles, In young birds with undeveloped wattles, injection and measurement are Design of Experinwnts difficult, and in older birds injections of (i) Birds immunized by infection. In the very thin but tough wattle dermis are not always truly intradermal (see Bush- Expts. l-4 three or more infected birds nell and Brandly 1931) but can be checked were tested at each time interval (usually of 2 weeks) from l-11 weeks after the iniby the appearance of blanching and a firm tial inoculation, by which time all the bleb. The hyperemic nature of the wattle also causes difficulties and precludes ob- birds had been used. In Expt. 5, birds servations other than measurements of were tested from Week 1 through to Week thickness. Readings were taken 20 min and 18 after inoculation, and in Expts. 5, 6, and 7, in order to obtain more ,data on at 4, 24, and 48 hr after .antigen injection. later responses,birds were tested at weekly Wattle thickness was measured with ordinary calipers (Expts. l-5) or, in the case intervaIs from Week 11 through to Week 18. Untreated coccidia-free controls were of the older birds, (Expts. 6 and 7) with dial g&uge calipers (“Pocotest A02,” Ca- also tested at each time interval. (ii) Birds injected with antigen in FCA: robronze Ltd., School Road, London W4). The r&&s, given in millimeters, represent Expts. 8-10. Birds were wattle-tested 2 the difference in thickness between the weeks after the last injection ‘of antigen and after the 4%hr reading, they were RHS and LHS wattles. In most experiments (l-7), birds were skin-tested once inoculated with oocysts orally and again only, but in experiments (S-10) where anti- wattle-tested 2 weeks later.
1: 1)
1:2)
0
26
30
0 0
9
0
10
10
Number of birds tested
tenella Oocyst
5 Tested approximately 2 weeks after last injection b Tested 2 weeks after oral inoculum. c Approximately 250 oocysts of H strain or 100,000 d Not done.
None
+ FCA
Saline
+ FCA
(Me)
Antigen
10
+ FCA
Saline
+ IAF,
injected
to Eimeriu
Antigen (00 + IAF, +FCA Saline + FCA None
(00
Antigen +FCA
Material
Responses
9
8
Experiment number
Wattle
oocysts
course
after
00
00
of TA
+ IAF)
of antigen.
(00
Test antigen
After
Antigen
TABLE
strain
I
given
0.1
0.2
0.1
orally
0
0.7
0.5
ND
2 weeks
0.2
0.4
0.4
0
after
and after After
Challenge
00
00
00 Mz IAF 00 MB IAF 00 Ms IAF
Test antigen
challenge
Infection
of antigen.
12 12 11 11 11 12
10 10
10
Number of birds tested
last injection
H TA H TA H TA
H
H
Oocyst inoculum strainc
in FCA
0.5
48
4
0.3 NDd
test
of reading (hr)
Time
in wattle (mm)
of Antigens
Difference thickness
of injection9
ip Injection
8-10)
0.5 0.7 0.6 0.5 0.1 0.2
0.1 0.1
0.8
ND
0.3
Time
1.2 1.0 1.4 0.9 0.3 0.3
0.5 0.5
1.0
ND
4
0.6 0.5 0.6 0.3 0.3 0.3
ND
0.1
0.6 0.4 0.2 1.1 0.2 0.1 1.0 0.5
48
test
in wattle (mm)
of reading @r)
Difference thickness
infectionb
(Expts.
g “M
I3
R M g
Eirneria
ten&a:
HWERSENSITIVITY 133 THE FOWL
RESULTS Immunization by Infection Eimeria tenella
with
The initial inoculum of E. tenella (TA) protected well against the pathogenic effects of E. tenella (H) in all experiments except Expt. 1 in which there was some blo’od in the feces after the first inoculum of E. ten&a (H). In Expts. 3-6 no oocysts were detected in the feces after the final inoculum of E. tenalla (H) (given at Week 4 ‘or 6) indicating that the birds had become completely immune; small numbers were detectable by salt flotation in Expts. 1 and 2. Immunity was again tested (Expt. 6 only) on Week 18 when four birds were given 50,000 moocysts of E. tenella (H); they did not show any clinical signs of disease but some soocysts were found in feces examined by salt flotation. Four control susceptible birds given the same inoculum suffered severe infection, with blood in the feces on Days 5 and 6 after oocyst inoculation, and large numbers of oocysts on Days 7 and 8. Attempted
Immunization
by Injection
of Antigens in FCA
The results of challenge with oocysts of E. tenella (H) or ( TA ) (to be given in detail elsewhere) were similar in all groups, whether injected with antigen in FCA, saline and FCA, or untreated. Thus no protection was afforded by the administration of these antigens. Skin Hypersensitivity ii) With Diferent
Antigens
Of the antigens tested, oocyst (00) produced in similarly treated birds the greatest responses and caused minimum reactions in coccidia-free animals. As this antigen was also the most easily obtainable it was used in the majority of the tests. Reactions to oocyst, merozoite, and IAF antigens in the same group of infected
133
(but other wise untreated) birds may be compared in Table I (Expt. 8, third group). In most cases delayed responses at 24 and 48 hr were similar, with a tendency to be greater at 48 hr. (ii) During and After Immunization by Infection Oocyst antigen was used throughout, but in different preparations in some of the experiments. The results for Expts. 1-4 (observations from Weeks 1 to 11) were pooled, as were those for Expt. 5, 6, and 7 (observations from Weeks 11 to 18); data for Expt. 5 are given separately (Weeks l18). Readings obtained from untreated control birds in all experiments were pooled (Fig. 1). Mean values obtained from readings at 20 min were low ( < 0.3 mm) and variable, but there were little or no differences between infected and control groups, especially from 11 to 18 weeks. Readings obtained at 24 and 48 hr were somewhat similar, hence the data for 24 hr are omitted from Fig. 1 in which the results obtained at 4 and 48 hr are given, together with the numbers of birds tested at each time interval. Reactions at 4 hr. A distinct pattern of response was obtained at this time interval. Values increased sharply to a peak at 4 to 7 weeks and then declined, again quite steeply, to low values at 11 weeks and thereafter. The highest mean values obtained represented ‘approximately a 100% increase in wattle thickness, but in a few individuals increases of between 150 and 200% were found, Reactions at 48 hr. Positive responses were obtained from the second week after infection, remaining at a fairly low to moderate value until Week 9 after which they increased quite considerably. From 11 to 18 weeks the values remained high but somewhat variable, while those obtained in the same birds at 20 min or 4 hr were particularly low. Maximum mean
134
M.
d 5
ELAINE
ROSE
1.0 -
.E
0.9
-
e 5 8 g
@7-
At 48hrs
3
0.5 -
0
Y 1
I
I
Y
3 Weeks
I 5
I
after
Y 7 initial
9
11
13
15
17
inoculum
FIG. 1. Dermal reactions 4 and 48 hr after injection of Einzeria tenella oocyst antigen in birds immunized by infection and in coccidia-free controls. Mean values are given, in millimeters, for differences in thickness between right hand (injected with antigen) and left hand (injected with saline) wattles. Numbers of birds tested at each time interval are given beside symbols. Immunized birds: pooled results of Expts. l-4, c]- - -0; results of Expt. 5, A. coccidia-free controls : pooled results, O-O; pooled results of Expts. 6 and 7, A----
o- -0. values represented again approximately a 100% increase in wattle thickness. The change in character Iof the response with time after the initial infection may be seen in the results (given in Fig. 2) for birds of Expt. 5 tested at Weeks 5 and 11. The histol.ogical features of the responses at 4 and 48 hr are shown in Fig. 3. The 4-hr reaction is one of acute inflammation
with edem.a and an infiltration of heterophi1 polymorphonuclear leucocytes. At 48 hr the reactions were characterized by an intense infiltration with small mononuclear cells, (iii) In Chickens Infected with Antigens in Freund’s Complete Ad/uvant Birds were wattle-tested approximately 2 weeks after the last antigen injection,
Eimeriu
&%?kz‘a:
HYPERSENSITIVITY
immediately preceding infection by oral inoculation with oocysts, ,and again 2 weeks later. The results are given in Table I. At the first test, wattle reactions were positive in all groups injected with antigen in FCA; 20-min values were negligible or low and the highest readings were obtained at 4 hr, but some delayed response was probably also present. Control groups injected with FCA and saline gave negative ,or negligible values although there was some indication of a possible delayed response at 48 hr in this group in Expt. 10. A challenge infection given to antigeninjected groups appeared to enhance the responses, particularly at 20 min, and in Expt. 10 groups previously injected with FCA and saline gave higher values than untreated groups. However, it is possible that these results were due to sensitization of the wattle by the previous testing. Transfer
of Skin Hypersensitivity
Limited attempts skin reactivity with (i) With serum.
were made to transfer serum and with cells. Two pools of serum
IN
THE
FOWL
were used for transfer experiments. One was ‘obtained from the birds of Expt. 4 bled 5 weeks after the initial inoculum of oocysts when the response at 4 hr was higher th,an at 48 hr, and the other was from three birds of Expt. 7 whose 4%hr responses were high and 4-hr responses very low (bled 14 weeks after the initial inoculum of oocysts ) . Five milliliters of the pooled serum were injected intraven’ously into each of three or five 3-week-old chickens, and 1 hr later antigen or saline was injected into the wattles. A control group of two birds was similarly treated after receiving serum from coccidia-free chickens. The results of wattle measurements made at 4, 24 and 48 hr later are given in Table II, together with the responses of the serum donors tested immediately before they were bled. In two of the three birds given the “early” serum, a low positive result was obtained at 4 hr; readings at 24 or 48 hr gave negligible or negative values. Thus, although only small numbers of animals were used, the 4 hr response did seem to be transferable with serum.
Week 11
Time. hours
after
135
injection
of wattle
FIG. 2. Values, mean and range, for dermal reactions (differences in thickness between wattle injected with antigen and wattle injected with saline) at different times after injection of Eimeria tenella oocyst antigen. Results from Expt. 5; tests carried out at 5 weeks (three birds) and at 11 weeks (three birds) after the initial inoculum of oocysts. Infected birds: n-----n; coccidia-free controls (three birds) : O- - -0.
136
M.
ELAINE
ROSE
&‘W??‘iU
tiXl&X
HYPERSENSITIVITY
Skin reactivity was absent at all test interin the birds which received the “late” serum (high delayed responses in donors) and in those which were given control serum. (ii) With cells. Suspensions of cecal tonsil and spleen cells were prepared from three birds killed 11 weeks after the initial inoculum of oocysts when the wattle responses were of the delayed type (mean difference in wattle thickness = 0.1, 0.6, ‘and 1.0 mm at 4, 24, and 48 hr, respectively) and from three control, coccidiafree birds (wattle responses negative). Each suspension (approx. 50 x lOa spleen cells or 2 X lo8 cecal tonsil cells) was injected intraperitoneally into a single 3week-old recipient and tests were done 4.25 hr later. No differences in thickness were apparent when the wattles were measured at 4, 24, and 48 hr; the responses seen in the donors had not been transferred to the recipients. vals
Correlation of Skin Hypersensitivity In Vitro Tests
with
(i) After immunization by infection. Pooled serum samples were obtained at weekly intervals from Expts. 2, 4, and 5 and tested for precipitating antibodies wth ‘oocyst antigen. Precipitin bands were found only with serum samples ‘obtained at Weeks 3, 4 ( Expt. 2, 4, and 5), and 5 (Expts. 2 and 4); all other samples gave negative results. Thus the presence of precipitating serum antibody coincided with the period of rapid increase and peak in the 4-hr response, but not with the decline phase. Tests for inhibition of macrophage migration in vitro in the presence of antigen were carried out on some ,of the animals whose wattle responses are summarized in
FIG. 3. Sections of wattles after injection of Eimeria weeks after initial inoculum eosin.
IN
THE
Fig. 1. In most cases peritoneal exudate (PE) cells were obtained from the birds immediately after the 48-hr wattle readings were taken, the antigens used being oocyst and merozoite. The results of these tests were highly variable; a small number of positive tests was observed, but the results did not correlate with wattle sensitivity. (ii) After injection with antigens in FCA. Serum samples were taken from the birds in all three experiments of this type after the course of antigen injections (immediately before oocyst inoculation) and again 2 weeks after ‘oocyst inoculation, and tested f.or the presence of precipitating antibodies with various antigens. The findings, given in Table III, are in fair agreement with the 4-hr wattle responses (Table I) in that (a) positive results were obtained in ‘antigen injected groups; these were enh,anced after infection and considerably stronger than those found in other groups, and (b) in Expt. 10 the group injected with FCA and saline which after infection gave higher wattle responses than the untreated groups, also had a greater proportion of birds with precipitating antibodies. Surprisingly, some sera of this group gave positive reactions before infection although there was no fecal evidence of extraneous infection. A few birds were used for MM1 tests after the output of oocysts from the infection with E. tenella (H) had ceased; results are given in Table III. In Expt. 10, leucocytes were used for some birds because of the difficulties ‘of obtaining large enough numbers of PE cells in older birds, especially after a series of intraperitoneal injections with FCA. A degree of inhibition was seen in a small number of infected (‘overall 5/15) birds, but there did
of control (A-D) and infected (E-H) chickens antigen or saline. Wattles injected 4 (E, of oocysts. Fixed in formal saline, stained with
tenelk
137
FOWL
taken 4 or 48 hr F) or 9 (G, H) hematoxylin and
138
M.
Transfer
of 4-hr
Skin
Serum Time of bleeding (weeks after initial oocyst inoculum)
ROSE
TABLE
II
to Eimeria
tenella
Oocyst
donors
Difference Time
Skin reactivity : in wattle thickness (mm) of reading test (hr) 24
48
5
0.8
0.4
0.5b
14
0.1
0.9
0.9c
0
Antigen
with
“Early”
Serum
Recipients”
4
0 Control coccidiafree a Each b Mean c Mean
Reactivity
ELAINE
0.1
0
Bird number
Difference Time
Skin reactivity: in wattle thickness (mm) of reading test (hr)
4
24
48
1 2 3 1 2 3 4 5 1
0.3 0.2 0 0 0 0 0 0.1 0
0.1 0.1 0 0 0 0.1 0 0 0
0.1 0 0
2
0
0
0
0 0 0
3-week-old bird given 5 ml of serum intravenously; antigen or saline injected 1 hr later. values from three birds tested at random from group of 20 which provided the serum pool. values from the three birds which provided the serum pool.
not seem to be any marked consistent differences between the groups. However, the numbers examined were small. DISCUSSION
The results of the tests for skin reactivity are expressed only in terms of wattle thickness, no other observations being possible because of the hyperemic nature of the wattIe. In highly reactive wattIes some temperature .difference may be apparent but this w.as not detected in the system used here. Hypersensitivity After Infection
Reactions
During
and
In the five experiments in which birds were tested at intervals during 11 weeks after an initial inoculum of Eimeria tenella (TA) followed by two or three inocula of E. tenella (H), the 4-hr reaction was the most intense and showed a ,definite rise and fall during the period of test. The pattern was similar to that previously found for serum cytophilic/opsonic antibody ac-
tivity (Rose 1974). Peak responses were noted at a time (Weeks 4-7) when protective activity has been demonstrated in serum (Rose and Long 1971) and, in experiments 2, 4, and 5, precipitating antibodies were found in the pooled serum samples at the time of the peak and in the 2 weeks before. In addition, 4-hr reactions appeared to be transferable with serum taken at Week 5. Thus this response was probably of the Arthus type. In the latter half of the test period the 4-hr response was negligible. Delayed reactions were also present from the second week after the first oocyst inoculum remaining at a moderate, fairly constant level until Week 9, after which they became much more marked. This increase coincided with the virtual disappearance of the early reactions. Unsuccessful attempts to transfer the delayed response with suspensions of spleen and cecal cells at Week 11 were probably due to histoincompatibility between donors and recipients, as was a previously reported failure to transfer delayed hypersensitivity
Eimeria
tenella:
HYPERSENSITIVITY
1976). A mild Arthus-type response was also present and this was transferable by serum, whereas the ,delayed response was transferred only with mesenteric lymph node cells. Calves infected with E. boois gave delayed reactions at 26 days after infection (Klesius, Kramer, Burger, and Malley 1975). The earlier development of predominantly delayed responses with these tw,o species, compared with E. teneZla in fowl, may be related to differences in immunogenicity. E. tenella is, with E. necatrix, one of the least immunogenic of the species parasitizing the chicken (Rose and Long 1962) and probably very poor in this respect in comparison with species parasitizing other hosts. The type of antigen used may also be a factor in determining the nature of the response obtained; in the work described here only soluble antigens prepared by physical means were used, whereas Klesius, Kramer, Burger, and Malley (1975) and Klesius, Kramer, and Frandsen (1976) used a particulate antigen prepared from unsporulated oocysts by TCA precipitation
reactions which otherwise fulfilled all the criteria applied to this form of immune response (see Warner, Ovary, and Kantor 1971). Histologically the reactions were typical of the delayed type and they were not transferred with serum (Table II). The results of MM1 tests did not provide any in vitro evidence of a CM1 response during this latter half of the test period, but the numbers examined were small, and it is possible that, as with leishmaniasis and toxoplasmosis in guinea pigs, positive results may be obtained only when the delayed skin reaction is very high (Tremonti and Walton 1970). Alternatively it is possible that as the cells for MM1 tests were obtained at the height of the wattle response (48 hr after injection of antigen), all the sensitized cells were mobilized to the wattle and therefore not available in the PE (see Granlund and Loan 1974). Responses of the 20-min type were low throughout the period of testing. In rabbits infected with E. s&due delayed reactions appeared to predominate earlier (Klesius, Kramer, and Frandsen TABLE Results of Tests for Precipitating Migration in Groups Injected Material
Experimerit number
injected
After
III
Serum Antibodies and for Inhibition with Eimeriu tenella Antigens in FCA course
139
IN THE FOWL
of injections
Precipitin test? Number positive with
dfter 00cgst strainb
of Macrophage (Expts. 8-10) challenge
infection
Precipitin test Number positive
:a with
test
MMITc antigens
-
8
Antigen (00 + IAF, + FCA Saline -I- FCA
IAF
Ma
00
O/10 o/10
IO/10 O/IO
10/10 O/IO
10
None, not infected Antigen (00 + IAF, 1: 1) +FCA Saline + FCA None None, not infected Antigen (Ma) + FCA Saline
+
FCA
None None,
not infected
MB
00
s/10 o/10 Q/Q
lO/lO o/10 Q/O
lO/lO 3/10 3/10
ND
lO/lO 4/10 3/10
Mz
00
l/2
o/2
O/l O/l O/l
l/l l/l O/l
1:2) H
None 9
IAF
X/IO
H
ND
o/10
26/29
23/29
O/28
3/28
H TA H TA H TA -
ND
0 Birds bled 2 weeks after last injection of antigen or oral inoculation of oocysts, respectively. 6 Approximately 250 oooysts of H strain or 100,000 oocysts of TA strain given orally 2 weeks e Tested 3-5.5 weeks after oooyst inoculation. Migration index below 0.8 = positive.
12/12 12/12 9/11 9/11 2/11 6/12
l/2 l/2 O/l O/l Q/6
10/12 12/12 6/11 4/11 o/11 l/12
o/2 after
last
inj&ion
of antigen.
140
M.
ELAINE
followed by pepsin digestion and acid extraction. Different ‘methods of preparing antigens from Trypanosonuz brucei have been shown to give different results for 4-hr relative to 24-hr reactions (Tizard and Soltys 1971), and in T. cruzi-infected rabbits the injection of soluble or particulate antigen fractions induced immediate ‘or delayed type responses, respectively (Teixeira and Santos-Buch 1975). The importance of the type of test antigen has als’o been shown in a Toxoplasma gondii test ,system (Lai, Tizard, Quinn, and Ingram 1975). It is therefore highly likely that the choice of test antigen has an important bearing on the results obtained and these ought to be evaluated accordingly. However, in the work described here, in which an increase in delayed response was found later in immunization, the same test antigen was used through’out. A late increase in delayed-type skin hypersensitivity has been noted in infections with ,other protozoa. In humans infected with T. gondii, DH to toxoplasmin occurs late in infection (Frenkel 1948), although in guinea pigs it is found from 1 week after (Krahenbuhl, Blazkovec, and Lysenko 1971). In T. brucei-infected rabbits, there is a change over from a 4-hr Arthus type ,of response found at 2 weeks to a delayed 24-hr type found not earlier than 3 weeks after the onset of infection (Tizard and Soltys 1971) and in monkeys infected with Plasmodium knowlesi, delayed hypersensitivity was found in only one chronically infected animal which was tested considerably later than any of the others (Phillips, Wolstencroft, Brown, Brown, and Dumonde 1970). However, in Leishmania enrietti-infected guinea pigs, DH developed early (Bryceson, Bray, Wolstencroft, and Dumonde 1970). Hypersensitivity Reactions of Antigens in ECA
After
Infection
Wattles of birds which had received injections of antigens in FCA became hypersensitive to the immunizing and other
ROSE
coccidian antigens. The reactions were most intense at 4 hr but in three of the four experiments ‘there appeared to be also a delayed response as wattle thickness was maintained throughout the test period. After a challenge infection the responses were enhanced and slightly greater than in the oontrol group injected with FCA alone. There was also a tendency for groups injected with FCA alone to be more reactive than previously untreated groups. This may have been due to the effect of adjuvant, or possibly to previous testing of the wattle to determine the preinfection responses. On the other hand, in two experiments precipitin responses were also higher in this group. Precipitating antibodies were constantly found in the sera of antigen injected birds, but results .of tests for MM1 gave rather inconclusive results. From the evidence presented here, delayed hypersensitivity is present during the earlier stages of the acquisitJon of immunity but becomes more marked later on when immediate responses have diminished. Immunity ‘of the birds to reinfection was checked at Week 4 when the 4-hr response was high and the 48-hr response lower, and at Week I8 when the 4-hr response was absent and the 4%hr response high. A substantial degree of immunity was found at b’oth times and is thus associated with both types of response in animals immunized by infection. However, birds injected with antigens in FCA and showing reactions predominantly of the immediate type (together with high levels of precipitating antibodies) were susceptible to subsequent infection. Similarly, rabbits to which DH had been transferred by TF prepared from animals infected with Eimeria stiedae were susceptible to infection (Klesius, Kramer, Burger, and Malley 1975). Thus, although the skins of immune animals are hypersensitive to injections of antigens, dermal reactivity, immediate or delayed, is not necessarily an indication of immunity to infection.
i!?ime?iia
t&U&a:
HYPERSENSITIVITY
ACKNOWLEDGMENTS The excellent technical assistance of Patricia Hesketh and Barbara Fisher is gratefully acknowledged. Thanks are also due to my colleague, Dr. P. L. Long, for help with photography, and to Drs. P H. Klesius, T. T. Kramer, and J. C. Frandsen, USDA-ARS-SR, Regional Parasite Research Laboratory, Auburn, Alabama, U.S.A., for showing me their manuscript before publication.
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IN THE FOWL
141
MORLTA, C., TEXJTSU~I, Y., AND SOEI(AWA, M. 1973. Migration inhibition test of splenic cells of chickens infected with Eimeria tenella. Journal of Parasitology 59, 199-200. PHILLIPS, R. S., WOLSTENCROFT, R. A., BROWN, I. N., BROWN, K. N., .~ND DUMONDE, D. C. 1970. Immunity to malaria. III. Possible occurrence immunity to of a cell-meriated Plasmodium knowlesi in chronically infected and Freund’s complete adjuvant-sensitised monkeys. Experimental Parasitology 28, 339355. ROSE, M. E. 1973. Immune responses to the Eimeria: Recent observations. In “Proceedings of the Symposium on Coccidia and Related Organisms,” pp. 92-118. University of Guelph, Ontario, Canada. Rose, M. E. 1974. Immune responses in infections with coccidia: Macrophage activity. Infection and Immunity 10, 862-871. ROSE, M. E., AND HESKETH, P. 1974. Fowl peritoneal exudate cells: Collection and use for the macrophage migration inhibition test. Asian Pathology 3, 297-366. ROSE, M. E., AND LONG, I?. L. 1962. Immunity to four species of Eimeria in fowls. Immunology 5, 79-92. ROSE, M. E., AND LONG, P. L. 1971. Immunity to coccidiosis: Protective effects of transferred serum and ceils investigated in chick embryos infected with Eimeria tenella. Parasitology 63, 299-313. RYLEY, J. F., AND BETTS, M. J. 1973. Chemotherapy of chicken coccidiosis. Advances in Pharmacology and Chemotherapy 11, 221-293. TEIXEIRA, A. R. L., AND SANTOS-BUCH, CA. 1975. The immunology of experimental Chagas disease. II. Delayed hypersensitivity to Trypanosoma cruzi antigens. Immunology 28, 401-410. TEARD, I. R., AND SOLTYS, M. A. 1971. Cellmediated hypersensitivity in rabbits infected with Trypanosoma brucei and Trypanosoma rhodesiense. Infection and Immunity 4, 67P 677. Tremonti, L., AND WALTON, B. C. 1970. Blast transformation and migration-inhibition in toxoplasmosis and leishmaniasis. American Journal of Tropical Medicine and Hygiene 19, 49-56. WAGEIYBACH, G. E., CHALLEY, J. R., AND BURNS, W. C. 1966. A method for purifying coccidian oocysts employing clorox and sulfuric aciddichromate solution. Journal of Parasitology 52, 1222. WARNER, N. L., OVARY, Z., AND KAXTOR, F. S. 1971. Delayed hypersensitivity reactions in normal and bursectomized chickens. Znternational Archives of Allergy and Applied lmmunology 40, 719-728.
