EFFECT OF IRRADIATION (GAMMA RAYS) ON THE BIOLOGY OF EIMERIA TENELLA OOCYSTS

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1 EFFECT OF IRRADIATION (GAMMA RAYS) ON THE BIOLOGY OF EIMERIA TENELLA OOCYSTS R.S. Bajwa, B.S. Gill To cite this version: R.S. Bajwa, B.S. Gill. EFFECT OF IRRADIATION (GAMMA RAYS) ON THE BIOLOGY OF EIMERIA TENELLA OOCYSTS. Annales de Recherches Vétérinaires, INRA Editions, 1977, 8 (2), pp <hal > HAL Id: hal Submitted on 1 Jan 1977 HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 EFFECT OF IRRADIATION (GAMMA RAYS) ON THE BIOLOGY OF EIMERIA TENELLA OOCYSTS R.S. BAJWA B.S. GILL College of Veterinary Science, Punjab Agricultural University, LUDHIANA , India Résumé EFFET DE L IRRADIATION (RAYONS GAMMA) SUR LA BIOLOGIE DES OOCYSTES D EIME RIA TENELLA. Les recherches ont porté sur la biologie de la descendance d oocystes d Eimeria tenella irradiés par des rayons gamma. L inoculum d oocystes sporulés était exposé à une irradiation gamma (6O Co) de 5 à 60 kr et administré par voie buccale à des poulets. Les oocystes excrétés par les poulets ont été récoltés et ont sporulé. Le taux de sporulation, la pathogénicité, l immunogénicité et le potentiel de reproduction de ces oocystes (descendance des oocystes irradiés) ont été comparés à ceux d oocystes non irradiés. On a observé que l augmentation de la dose d irradiation entraîne une baisse progressive de la pathogénicité de la suspension d oocystes. Les oocystes exposés à 30 et 40 kr provoquaient seulement une infestation légère, tandis que ceux exposés à 50 kr ou plus étaient non infestants. Aucune différence dans la pathogénicité, l immunogénicité et le potentiel de reproduction n a été observée entre des oocystes non irradiés et la descendance des oocystes irradiés. On en conclut que l effet de l irradiation était limité à l inoculum exposé au rayonnement, et n était pas transmissible à sa descendance. Introduction Exposure of oocysts of Eimeria tenella to ultraviolet rays (Fish, 1932), Xrays (Albanese and Smetana, 1934 ; Waxler, 1941 ; Sibalic, 1970) and gamma rays (Baldelli et al., 1966 a, b, c, d ; Bajwa and Gill, 1975), and of E. necatrix (Singh and Gill, 1975) and E. acervulina (Ali et al., 1972) to gamma rays have been reported to result in decreased infectivity. Waxler (1941), Baldelli et al. (1966 d) and Ali et al. (1972) were of the view that the irradiation attenuated the parasite whereas Fitzgerald (1968), Bajwa and Gill (1975) and Singh and Gill (1975) expressed the opinion that the irradiation did not attenuate the parasite but only had the dilution effect on the oocyst suspension by reducing the number of viable oocysts/sporozoites in it. The present authors studied the sporulation rate, pathogenicity, immunogenicity and reproduction potential of the progeny of the irradiated oocysts of E. tenella with a view to provide more information on the controversial topic of the effect of irradiation on

3 oocysts. paper. The results are presented in this Materials and methods The chicks Dayold male WhiteLeghorn chicks reared in battery brooders with raised wirebottomed floors, were used. The infected and uninfected chicks were maintained in separate noncommunicating rooms, and were handled by separate attendants. Before giving the experimental infection, freedom of the chicks from adventitious coccidial infection was ascertained by examining faeces by salt floatation technique for oocysts. Chicks of two weeks of age, were used in the experiments. The parasite Singleline strain of E. tenella was employed. Suspension of oocysts were prepared by harvesting caeca on eighth day of the infection, the mucosa scraped with a scalpel, and the scrapings and the caecal contents tearedina a Waring blender containing 2.5 per cent potassium dichromate solution. The suspension was poured into Petri dishes to a depth of about 5 to 10 mm and allowed to sporulate at 27 to 30 C. Before feeding to chicks, the oocysts were washed free of the potassium dichromate and resuspended in water, and the number of the oocysts in the suspension was counted with a McMaster chamber. Irradiation Universal bottles made of neutral glass containing the requisite number of the sporulated oocysts in 10 ml suspension in distilled water, were irradiated in a Gamma Cell (Bhabha Atomic Research Centre, Bombay, India) using 6OCO as the source or radiation. The irradiated oocysts were kept at room temperature for two days, and tested for infectivity. Design of the experiment Susceptible chicks were infected by placing 1 ml of the oocysts suspension in the pharynx. Mortality and bleeding, if any, were noted. Half the number of chicks was sacrificed on the sixth day of the infection and lesions scored according to the method described by Johnson and Reid (1970). Faeces of the remaining chicks were collected every 24 hours from the seventh to the 14th day and oocysts counted following the method of Long and Rowell (1958). On the 14th day, all the surviving chicks were sacrificed, caeca harvested and torn in a Waring blender and suspended in water. The oocysts in the suspension were counted. Adding up the two counts gave the total number of oocysts produced. The rate of sporulation of the oocysts was ascertained as follows. The oocysts were separated from the faecal material by salt floatation technique and suspended in 2.5 per cent potassium dichromate solution. The suspension was poured into Petri dishes to a depth of about 5 to 10 mm and allowed to sporulate at 27 to 30 &dquo;c. The suspension was frequently disturbed by blowing air into it through a sterile Pasteur pipette to facilitate sporulation. The oocysts were examined under the microscope after 18, 36, 48, 60 and 72 hours, and the number of sporulated oocysts in 200 oocysts examined, was counted. Pathogenicity of the progeny of irradiated oocysts was tested by the study of the infections caused by 50,000 oocysts per chick as described above. lmmunogenicity of the oocysts produced by the irradiated oocysts was evaluated as follows. Chicks recovered from the initial or immunizing infections by 1,000 and 5,000 oocysts which were the progeny of the irradiated oocysts, were challenged with 50,000 oocysts per chick on the 15th day. Blood in faeces, and mortality, if any, were noted. Half the number of chicks was sacrificed on the sixth day of the challenge and lesions scored. The number of oocysts present in the faeces of the remaining chicks on the seventh to the ninth day, and in the caeca of the chicks sacrificed on the ninth day was determined as described above. The sum of the two counts gave the total number of oocysts produced as a result of the challenge of infection. Effect of the irradiation on the reproduction potential of the oocysts was assessed by ascertaining the reproduction index of the progeny of the irradiated oocysts. The reproduction index was calculated by dividing the total number of oocysts produced per chick by the number of oocysts in the inoculum. Adequate controls i.e. the infections produced by the unirradiated oocysts were kept in all the experiments.

4 Results The observations on the infections produced by 50,000 oocysts exposed to different levels of the irradiation are presented in Table 1. The data on the effects of the irradiation on pathogenicity, and on immunogenicity and reproduction potential of the oodysts which were the. progeny, of the irradiated oocysts are summarised in Tables 2 and 3, respectively. Reference to Table 1 reveals that increase in the irradiation dose caused progressive decrease in the pathogenicity of the oocyst suspension. The oocysts exposed to 50 kr and above, were noninfective as evident from the lack of lesions and nonproduction of oocysts. Chicks infected with the oocysts exposed to 30 and 40 kr developed only very mild infections as evident from the absence of the lesions and the production of a few number.of oocysts detectable only on salt floatation of the faeces. There was no difference in sporulation of the oocysts derived from the irradiated and unirradiated oocysts, as 85 to 93 % of them

5 sporulated in 24 to 60 hours in all cases. Infections produced by oocysts derived from the unirradiated or irradiated parent oocysts were similar as evident from the insignificant differences in mortality rates, lesion scores and the total oocyst production (Tables 2 and 3) or the grade of immunity induced by 1,000 and 5,000 oocysts (Table 3). Discussion Radiation dose was found to bear an inverse relationship with infectivity of the sporulated oocysts as the increase in the dose of irradiation of the oocyst suspension, resulted in progressively less severe infections as evident from the decreased amount of blood in faeces (Table 1). Exposure to 50 kr or above, rendered the oocysts noninfective. Oocysts exposed to 30 and 40 kr produced only mild infections. These results are in conformity with those reported earlier by Bajwa and Gill (1975) on E. tenella, and by Singh and Gill (1975) on E. necatrix. The increase in the number of the oocysts produced by the chicks infected by oocysts irradiated up to 20 kr (Table 1) was possibly due to the progressive decrease in the crowding effect as it has been shown previously (Bajwa and Gill, 1975) that irradiation decreased the number of the viable sporocysts/sporozoites in the suspension. It is wellknown that severe infection by E. tenella causes profuse bleeding due to extensive destruction of the caecal mucosa, consequent greater loss of the second generation merozoites and production of smaller number of oocysts. The decreased oocyst output by inocula irradiated at 25 kr and above was due to the increasing nonviability of the sporozoites/ oocysts. The irradiation of the sporulated oocysts

6 Amer. the J. modified the character of the immediate infection mentioned above. But the irradiation did not have any effect on progeny oocysts which set up infection comparable to those caused by unirradiated oocysts. It is concluded, therefore, that the effect of irradiation was only limited to the inoculum exposed to it, and that it was not transmissible to the progeny of the irradiated oocysts. This conclusion further strengthens the earlier observations of Fitzgerald (1968), Bajwa and Gill (1975) and Singh and Gill (1975) that irradiation reduced the number of viable oocysts/sporozoites in the suspension. (Accepted for publication, March 1977.) Summary Effect of gamma rays on the biology of the progeny of the irradiated Eimeria tenella oocysts was investigated. The parent inoculum of sporulated oocysts was exposed to 5 to 60 kr (gamma rays). These oocysts were fed to chicks. The oocysts voided by the chicks were collected and sporulated. The sporulation rate, pathogenicity, immunogeni i city and reproduction potential of these oocysts progeny of the irradiated oocysts were compared with those of the unirradiated oocysts. It was observed that increase of irradiation dose caused progressive decrease in the pathogenicity of the oocyst suspension. The oocysts exposed to 30 and 40 kr produced only mild infections whereas those exposed to 50 kr and above, were noninfective. No difference in pathogenicity, immunogenicity and reproduction potential of unirradiated oocysts and the oocysts progeny of the irradiated oocysts, was seen. It was concluded, therefore, that the effect of irradiation was limited to the inoculum exposed to it, and was not transmissible to the progeny of the irradiated oocysts. R9fer ences ALBANESE A.A. and SMETANA H., Studies on the effect of Xrays on the pathogenicity of Eimeria tenella. J. Hyg., 26, ALI N.A., BINNERTS W.T. and KLIMES B., Immunization by irradiated Eimeria acervulina. J. Protozool., 19, BAJWA R.S. and GILL B.S Effect of irradiation (Gammarays) on oocysts of Eimeria tenella. Ann. Soc. belge. Med, trop., 55, BALDELLI B., ASDRUBALI G., BEGLIOMINI A., FRESCURA T. and MASSA D., 1966a. Studio degli effetti delle radiazioni gamma sui coccidi de polli. I. Effetti delle radiazioni gamma sulla sporulazione di oocisti di Eimeria tenella. Soc. ltal. Sci. Vet., 20, BALDELLI B., ASDRUBALI G., BEGLIOMINI A., FRESCURA T. and MASSA D b. Studio degli effetti delle radiazioni gamma sui coccldi dei polli. II. Potere infettante e immunizzante di oocisti di Eimeria tenella irradiate prima delia sporulazione. Soc. ltal. Sci. Vet., 20, BALDELLI B., ASDRUBALI G., BEGLIOMINI A., FRESCURA T. and MASSA D., 1966c. Studio degli effetti delle radiazioni gamma sui coccidi dei polli. Ill. Potere infettante di oocisti di Eimeria tenella irradiate dopo la sporulazione. Soc. ltal. Sci. Vet., BALDELLI B., ASDRUBALI G., BEGLFOMINI A., FRESCURA T. and 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. Soc. ltal. Sci. Vet., 20, FISH FF., Some factors in the control of coccidiosis of poultry. Am. Vet. Med. Ass., FITZGERALD P.R., Effects of ionizing radiation from cobalt 60 on oocysts of Eimeria bovis. J. Parasitol., 54,

7 J. Symposium Lab. JOHNSON J. and REID W.M., Anticoccidial drugs; lesion scoring techniques in battery and floorpen experiments with chickens. Exp. Parasitol., 28, 306. LONG P.L. and ROWEL J.G., Counting oocysts of chicken coccidia. Pract. 7, SIBALIC S., TOMANOVIC B. and MOVSESIJAN M., The effect of irradiating the infective and noninfective oocysts of Eimeria tenella with X and gamma rays on the possibility of their further development. Peradarski dani, Mostar SINGH J. and GILL B.S., Effect of gammairradiation on oocysts of Eimeria necatrix. Parasitology, 71, WAXLER S.H., Immunization against caecal coccidiosis in chickens by the use of Xray attenuated oocysts. Am. Vet. Med. Ass., 99, 4815.