Parasitology (1975), 71, 117-124 117

Effect of gamma-irradiation on oocysts of Eimeria necatrix

JASMER SINGH and B. S. GILL
Punjab Agricultural University, Ludhiana, Punjab, India

(Received 30 January 1975)

SUMMARY
Effect of gamma radiation on oocysts of Eimeria necatrix was investi-
gated. It was observed that oocysts exposed to 200 kR or above did not
sporulate. Irradiation at 10-150 kR caused a progressive decrease in
sporulation. Irradiation affected normal development of unsporulated
oocysts as the zygote protoplasm divided into unequal masses or was
shattered into granules.
Increase in the intensity of irradiation of sporulated oocysts resulted in
the progressive decrease in severity of the resultant infections in chicks
and their effects - mortality, type of lesions developed, total oocyst
production and immunity produced - were comparable with infections
induced by decreasing the number of unirradiated oocysts. Infection
produced by 1000 unirradiated oocysts was comparable with that result-
ing from 50 000 oocysts irradiated at 25 kR. Infection obtained with
20 000 unexposed oocysts approximated to that produced by 50 000
oocysts irradiated at 2-5 kR. It was concluded that irradiation abolished
infectivity of the oocysts/sporozoites rather than bringing about attenu-
ation of the parasite.

INTRODUCTION
Exposure of oocysts of E. tenella to ultra-violet rays (Fish, 1932), X-rays
(Albanese & Smetana, 1937; Waxier, 1941; Sibalic, Tomanovic & Movsesijan, 1970)
or gamma rays (Baldelli, Asdrubali, Begliomini, Frescura & Massa, 1966a, b, c;
Sibalic et al. 1970) caused progressive loss of infectivity. A similar effect of gamma
rays on E. acervulinaw&s reported by Ali, Binnerts & Klimes (1972), who showed
that the inoculation of irradiated oocysts caused mild infection in chicks with
immunity to reinfection. On the basis of such observations, some authors (Waxier,
1941; Baldelli et al. 1966rf) concluded that irradiation brought about attenuation
of the coccidia. However, the same effects, namely mild infection and immunity,
could have been produced by infection with smaller numbers of unirradiated
oocysts. Fitzgerald (1968) concluded that irradiation killed sporozoites and reduced
the number of viable oocysts in the irradiated suspension. Ali et al. (1972) put
forward another view stating that irradiation probably killed or at least rendered
sporozoites unable to multiply and that intermediate doses of irradiation might
affect vitality of coccidia, leaving immunogenicity intact.
The present investigation was undertaken to elucidate the effect of irradiation
(gamma rays) on E. necatrix. Results are presented in this paper.
118 J. SINGH AND B. S. GILL

MATERIALS AND METHODS

The chicks
Day-old male White-Leghorn chicks were reared in battery brooders with
raised wire-netting floors. The brooders were electrically heated in winter. After
the termination of an experiment, the brooders were thoroughly scrubbed, washed
and dried. These were then singed with a blow lamp. The chicks were fed standard
chick mash.
Infected and uninfected chicks were maintained in separate non-communicating
rooms and were handled by separate attendants. Before the experimental in-
fection, freedom of chicks from adventitious coccidial infection was ascertained by
examination of faeces by the centrifugal-flotation technique using saturated com-
mon salt solution as the levitating medium. Chicks of 2 weeks of age were used in
the experiments.

The parasite
A single-line strain of E. necatrix, isolated from a natural outbreak and propa-
gated in susceptible chicks, was employed. The same strain of the parasite was
used throughout the investigation. The infected chicks were sacrificed on the
8th day and their caeca harvested. The caecal pouches were slit open longitudin-
ally and the contents along with scraped mucous membrane were collected. Such
collections from several chicks were suspended in 2-5 % potassium-dichromate
solution. The suspension was torn in a Waring blender and strained through a set
of sieves (20 meshes/cm). The suspension was poured into Petri dishes to a depth
of about 5-10 mm and allowed to sporulate at room temperature (25-30 °C). The
suspension was frequently disturbed by blowing air into it through a sterile
Pasteur pipette to facilitate sporulation. When fully sporulated, in 2-3 days, the
suspension was poured into a bottle and stored at +5 °C approximately.

Irradiation
Oocysts were washed free of potassium dichromate, resuspended in water and
irradiated. Ten ml of suspensions containing similar numbers of oocysts were
placed in Universal bottles made of neutral glass and irradiated. Irradiation was
carried out in Gamma Cell (Atomic Energy Agency of Canada, Ltd) using ^Co
as the source of radiation.
Both the freshly harvested, unsporulated oocysts and sporulated oocysts im-
mediately after sporulation was complete were irradiated. Potassium dichromate
solution was added to the unsporulated oocysts after irradiation and the suspension
poured as thin layer in Petri dishes which were frequently disturbed to facilitate
sporulation. The Petri dishes were kept at room temperature (25-30 °C). The final
irradiated and sporulated or sporulated and irradiated oocysts were tested for
infectivity or immunogenicity 1-2 days after completion of the process.
The effect of irradiation on the morphology and infectivity of the oocysts was
observed.
 Effect of radiation on E. necatrix 119

Design of experiment
Any change in morphology of the oocysts due to irradiation and in the sporula-
tion of oocysts irradiated during the unsporulated stage was noted by examining
the oocysts under a research microscope every 2-4 h.
The infectivity of the washed sporulated oocysts irradiated in the unsporulated
stage was tested by placing the suspension (about 0-5 ml) in the pharynx of chicks.
Half the number of chicks were killed on the 6th day of the infection and severity
of the lesions scored using a scale similar to that described by Johnson & Reid
(1970). Oocysts in faeces of the remaining chicks were counted from the 7th to the
9th day. On the 9th day all the surviving chicks were killed, their caeca harvested
and torn in a Waring blender and suspended in water. The number of oocysts
present in the suspension was counted. Adding up the two counts gave the total
number of oocytes produced.
The infectivity of the washed oocysts, irradiated in the unsporulated stage and
then allowed to sporulate, was tested as described above except that the remaining
half of the chicks on the 6th day were not killed. Faeces of these chicks were
collected every 24 h from the 7th to the 14th day of the infection and the total
number of oocysts voided by a chick was determined following the method de-
scribed by Brackett & Bliznick (1949).
In experiments evaluating immunity, the chicks survived from the initial or
immunizing infection were challenged with 50 000 oocysts per chick. The oocysts
were less than 14 days old after sporulation. The challenge infection was given on
the 15th day of the initial infection. Presence of blood in faeces and mortality, if
any, were noted. Half of the chicks were killed on the 6th day of the challenge and
lesions scored. The number of oocysts present in the faeces of the remaining chicks
on the 7th to the 9th day and in the caeca of the chicks killed on the 9th day was
determined as described above. The total of the two counts gave the number of
oocysts produced.
In order to compare the infections produced by inocula containing 50 000 sporu-
lated oocysts exposed to different levels of irradiation, with those produced by
varying numbers of unirradiated sporulated oocysts, groups of chicks of comparable
age were infected with increasing number of the latter oocysts. The characters of
the resultant infections studied were: blood in faeces, mortality, average lesion
score of half the number of chicks killed on the 6th day and total number of
oocysts produced from the 7th to the 14th day. The survivors were challenged on
the 15th day with 50 000 unirradiated sporulated oocysts and the above-noted
characters of the infection studied.

RESULTS
Effect of irradiation on unsporulated oocysts
The effect of irradiation on the morphology and subsequent sporulation and
infectivity of freshly voided unsporulated oocysts has been summarized in
Table 1.
It was observed that oocysts exposed to 200 kR and above did not sporulate.
 120 J. SINGH AND B. S. GILL

Table 1. Showing the infectivity of oocysts sporulated after irradiation

(Eight chicks were present in each group, and each chick received
50000 oocysts.)
Total oocyst
Average production
lesion from the 7th
Oocysts Blood score to 9th day
Irradiation sporulated in Mortality on the (million
(kR) (W
\ ml\ faeces (%) 6th day oocysts/chick)
0 92 + 75 400 2-40
10 90 + 75 4-00 2-50
25 81 + 50 3-67 2-25
50 58 + 25 2-67 3-90
75 30 + 25 2-33 4-10
100 17 + 0 1 5-26
150 10 Nil 0 1 5-80
175 8 Nil 0 0 500
200 0 Nil 0 0 0-00
300 0 Nil 0 0 0-00

Exposure to 10 kR did not have any appreciable effect on the oocysts as these
sporulated normally and produced infection in chicks comparable to that caused
by the same number of unirradiated sporulated oocysts. Further increases in the
dose of radiation resulted in progressive decrease in sporulation rate and severity
of the infection produced in chicks. Ninety, 81, 58, 30, 17, 10 and 8% of oocysts
irradiated at 10, 25, 50, 75, 100, 150 and 175 kR, respectively, sporulated as
compared with 92 % sporulation of the unirradiated oocysts. The inoculum exposed
to 100 kR produced clinically recognizable infection, and that irradiated at 150 kR
produced the lesion, i.e. haemorrhagic spots on the caecal mucous membrane but
no blood appeared in the faeces. Oocysts exposed to 175 kR set up infection, as
evident from the production of oocysts, but did not produce the lesion.
Exposure greater than 10 kR progressively induced aberrations in the morpho-
logy of oocysts, namely zygote mass shattered into granules or development not
proceeding beyond division of the zygote protoplasm into 2 or 4 equal or 2, 3 or 4
unequal masses, were observed.
The total number of oocysts produced by chicks infected with the irradiated
oocysts increased with the increase in the dose of irradiation. This increased oocyst
production could have been due to progressive decrease in the ' crowding effect'
as increasing intensity of irradiation resulted in decreasing number of viable
oocysts/sporozoites in the inocula. Severe infection causes extensive haemorrhage
and greater loss of second generation sporozoites and, consequently, numerically
inferior gametogony resulting in production of fewer oocysts.

Effect of irradiation on sporulated oocysts

Results of the infection produced by 50 000 oocysts exposed to different levels of
irradiation after sporulation and the resultant immunity are presented in Table 2.
Similarly, results of the infection due to varying number of unirradiated oocysts
and the ensuing immunity are summarized in Table 3.
 Table 2. The nature of the immunizing and challenge infections
(The chicks, 20 in each group, were infected with 50 000 oocysts irradiated after sporulation, and challenged on the 5th day
of the initial infection with 50 000 unirradiated oocysts.)
Immunizing infection Challenge

Average Total Average Total oocyst •at

lesion oocyst lesion production
Blood score production Blood score 7th to 9th day
Irradiation in Morta on 6th (million oocysts/ in Mortf on 6th (million
/ 0/ day oocysts/chick)
(kR) faeces V /O day chick) faeces (°A I
+ 75 3-88 3-35 Nil 0 0 0
2-5 + 20 2-83 4-60 Nil 0 0 0
5 4. 5 2-20 6-60 Nil 0 0 0
10 4_ 0 1-80 5-50 Nil 0 0 0
15 4. 0 1-30 4-95 Nil 0 0 0
20 Trace 0 110 3-65 Nil 0 0 0
25 Nil 0 0-40 1-00 Trace 0 1-00 0-30
30 Nil 0 0 * + 30 317 2'00
40 Nil 0 0 0 + 60 3-75 2-80 I
50 Nil 0 0 0 4- 70 3'88 2-20
60 Nil 0 0 0 4. 70 3-88 218
0 _ 4_ 70 3-90 2-28
(fresh
control)
Oocysts detectable only on salt flotation.
Table 3. The nature of the infections initiated by varying doses of unirradiated oocysts and of the challenge infection
(The challenge inoculum consisted of 50 000 oocysts per chick and was given on the 15th day of the immunizing infection.
Each group contained initially 50 chicks.)
Immunizing infection Challenge

Total oocyst Total oocyst

production Average production «H
Average from 7th to lesion from 7th to Ul
M
lesion 14th day Blood score on 9th day
Inoculum Blood in Mortality score on (million in Mortality 6th day of (million o
(oocysts) faeces (%) 6th day oocysts/chick) faeces to/ \ challenge oocysts/chick) M
1 000 Trace 0 0-60 2-40 (+) 0 0-80 0-24 %
5 000 + 0 1-60 515 Nil 0 0 0 H
10 000 + 10 2-55 5-00 Nil 0 0 0 td
20 000 20 2-92 4-20 Nil 0 0 0
30 000 + 35 3-69 3-80 Nil 0 0 0
40 000 55 3-80 3-50 Nil 0 0 0
h
M

50 000 75 3-88 3-35 Nil 0 0 0

50 000 _ + 70 3-90 2-28
(Fresh
control)
 Effect of radiation on E. necatrix 123
It was observed that increasing irradiation of the sporulated oocysts produced
infections of progressively decreasing severity (Table 2). Exposure to 40 kR and
more rendered the oocysts progressively less infective because chicks given these
oocysts failed to show evidence of the infection and were fully susceptible to the
challenge.
Chicks given oocysts irradiated at 2-5-20 kR developed infections of decreasing
severity and proved to be fully resistant to reinfection (Table 2). Oocysts exposed
at 25 kR produced mild infections which induced partial but considerable immu-
nity as reinfections did not cause mortality but only a few lesions and oocysts
were produced. Infections produced by the oocysts irradiated at 30 kR failed to
produce macroscopically detectable lesions. A few oocysts were detected only on
salt flotation of the faeces. The resultant immunity was of poor grade.
The increase in total oocyst production by the chicks infected with the irradiated
oocysts (Table 2), with the increase of exposure up to 10 kR, was probably due to
lessening of the ' crowding effect'. With further increase in the dose of irradiation,
the total oocyst production progressively decreased. This effect appeared to be
due to the increasing non-viability of the oocysts/sporozoites brought about by
the irradiation.
Inocula containing 5000, 10 000, 20 000 and 50 000 unirradiated oocysts pro-
duced infections of increasing severity and induced development of solid immunity
(Table 3). Infection with 1000 oocysts produced immunity of good grade.
From the infections produced by irradiated and unirradiated oocysts, it is
apparent that the infection produced by 50 000 oocysts irradiated at 25 kR was
somewhat comparable with that resulting from 1000 unirradiated oocysts and the
grade of immunity developed was also the same in the two cases. Further, infection
with 50 000 oocysts irradiated at 2-5 kR was comparable, in all respects, with that
produced by 20 000 unirradiated oocysts.

DISCUSSION
The investigation revealed that unsporulated oocysts of E. necatrix were less
susceptible to the action of irradiation than sporulated oocysts, as the minimum
exposure required to abolish the biological activity of sporulation, in the case of the
former, and viability, in the case of the latter type of oocysts, was 200 kR and
40 kR, respectively. Baldelli et al. (19666, c) and Fitzgerald (1968) reported similar
findings with E. tenella and E. bovis, respectively. The morphological aberrations
resulting from irradiation of unsporulated oocysts were similar to those observed
in E. stiedae (Fitzgerald, 1968).
Irradiation of sporulated oocysts resulted in progressively less severe infections
in chicks. The infections were comparable in all respects with those ensuing from
inoculation with a lower number of unirradiated oocysts. Lesions produced by
E. necatrix are caused by maturation of the second-generation schizonts. Oocyst
formation is the terminal stage of endogenous development, which has an ap-
proximately quantitative relationship with the number of oocysts initiating
the infection. Any modification in the endogenous cycle of the parasite caused by
irradiation would be reflected in the mortality of host, type of lesions developed
 124 J. SINGH AND B. S. GILL

(i.e. lesion score), number of oocysts produced and the resultant immunity. In the
present case, infection produced by 50 000 oocysts irradiated at 2-5 kR was
comparable with that resulting from 20 000 unirradiated oocysts. As the quanti-
tative effect of infection in the two cases was the same, it was inferred that
irradiation reduced the number of viable oocysts/sporozoites from 50 000 oocysts
to 20 000 unirradiated oocysts/equivalent number of sporozoites. Similarly, ex-
posure of 50 000 oocysts at 2-5 kR appears to have reduced the number of viable
oocysts/sporozoites to 1000 oocysts.
The investigation supported the view that irradiation reduced the number of
viable oocysts/sporozoites rather than brought about attenuation of the parasite
in the usual sense of the term. No additional advantage accrued from irradiation
of oocysts as the same immunity could be achieved by employing smaller numbers
of unirradiated oocysts.
The senior author (J.S.) is thankful to the Punjab Agricultural University for
the award of the Merit Scholarship in Parasitology.

REFERENCES
ALBANESE, A. A. & SMETANA, H. (1937). Studies on the effect of X-rays on the pathogenicity
of Eimeria tenella. American Journal of Hygiene 26, 27-39.
ALI, N. A., BINNEBTS, W. T. & KLIMES, B. (1972). Immunization by irradiated Eimeria
acervidina. Journal of Protozoology 19, 177-80.
B A L D E L U , H . , ASDBUBALI, G., BEGLIOHINI, A., FBESCUBA, T. & MASSA, D. (1966a). Studio
degli effetti delle radiazioni gamma sui coccidi dei polli. I . Effetti delle riadiazioni gamma
sulla sporulazione di oocisti di Eimeria tenella. Atti delta Societa Italiana delle Scienze
Veterinarie 20, 701-4.
B A L D E L U , B . , ASDBUBALI, G., BEGLIOMINI, A., FBESCURA, T. & MASSA, D. (1966b). Studio
degli effetti delle radiazioni gamma sui coccidi dei polli. I I . Potere infettante e immuni-
zzante di oocisti di Eimeria tenella irradiate prima della sporulazione. Atti della Societa
Italiana delle Scienze Veterinarie 20, 705-8.
BALDEIXI, B., ASDBUBAM, C , BEGLIOMINI, A., FBESCUBA, T. & MASSA, D . (1966C). Studio
degli effetti delle radiazioni gamma sui coccidi dei pelli. I I I . Potere infettante di oocisti di
Eimeria tenella irradiate dopo la sporulazione. Atti della Societa Italiana delle Scienze
Veterinarie 20, 709-12.
BALDELLI, B., ASDBUBALI, G., BEGLIOMINI, A., FBESCUBA, T. & MASSA, D. (1966d). Studio
degli effetti delle radiazioni gamma sui coccidi dei polli. IV. Potere immunizzante di oocisti
di Eimeria tenella irradiate dopo la sporulazione. Atti della Societa Italian delle Scienze
Veterinarie 20, 713-6.
BBACKETT, S. & BLIZNICK, A. (1949). The effect of small doses of drugs on oocyst production
of infections with Eimeria tenella. Annals of the New York Academy of Sciences 52, 595-610.
FISH, F . F . (1932). Some factors in the control of eoccidiosis of poultry. Journal of the American
Veterinary Medical Association 80, 543-59.
FITZGEBALD, P . R. (1968). Effects of ionizing radiation from cobalt-60 on oocysts of Eimeria
bovis. Journal of Parasitology 54, 233-40.
JOHNSON, J . & R E I D , W. M. (1970). Anticoccidial drugs; lesion scoring techniques in battery
and floor-pen experiments with chickens. Experimental Parasitology 28, 30-6.
SIBALIC, S., TOMANOVIC, B . & MOVSESIJAN, M. (1970). The effect of irradiating the infective
and non-infective oocysts of Eimeria tenella with X- and gamma rays on the possibility of
their further development. Symposium Peradarski dani, Mostar, 188-95.
WAXLEB, S . H . (1941). Immunization against caecal eoccidiosis in chickens by the use of
X-ray attenuated oocysts. Journal of the American Veterinary Medical Association 99,
481-5.
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