EXPERIMENTAL

71, 100-106 (1990)

PARASITOLOGY

Schistosoma YOU-EN

japonicum: Applicable SHI,*

CHANG-FU

*Department of Parasitology, The People’s Republic Im Neuenheimer

of China,

SHI, Y.-E.,

C.-F.,

JIANG,

An Ultraviolet-Attenuated Cercarial in the Field for Water Buffaloes JIANG,* JIA-JUN HAN,* YONG-LONG ANDREAS RuPPELt

Vaccine

LI,* AND

To&i

Medical University, Hang-Kong-Lu 13, Wuhan, Hubei Province, and tlnstitute of Tropical Hygiene, Universi@ of Heidelberg, Feld 324, D-6900 Heidelberg, Federal Republic of Germany HAN,

J.-J.,

LI,

Y.-L.,

AND RUPPEL,

A. 1990.

Schistosoma

cercarial vaccine applicable in the field for water buffaloes. Experimentai Parasitoiogy 71, 100-106. Water buffaloes were vaccinated three times with 10,000 Schistosoma japonicum cercariae irradiated with ultraviolet (uv) light at a dose of 400 pW . m&/cm*. The irradiation was performed with cheap, simple, and portable equipment in a rural area of Hubei Province (People’s Republic of China). A challenge infection of 1000 untreated cercariae was given to six vaccinated and six naive control buffaloes, while two vaccinated animals were not challenged. The experiment was terminated 6 weeks after the challenge. Control animals had lost body weight and harbored a mean of 110 worms and 37 eggs per gram of liver. The vaccinated animals gained weight after the challenge and developed 8% resistance to infection with S. japonicum. Since schistosomiasis japonica is nowadays transmitted in China predominantly by domestic livestock, a uv-attenuated cercarial vaccine for bovines may contribute to the control of this disease. 0 1990 Academic Rcss, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Schistosoma japonicum; Trematode, parasitic; Cercariae, attenuated; Ultraviolet (UV) light; Vaccination; Bubalus bubalis, water buffalo; Resistance to infection; Schistosomiasis, Control.

japonicum:

An ultraviolet-attenuated

Schistosomiasis is a major parasitic disease in China. Although the prevalence has been greatly reduced during recent decades (Cheng 1971; Mao and Shao 1982; Chen 1988), infections with Schistosoma juponicum still represent a serious problem in several provinces, in particular along the Yangtse River. In Hubei Province, schistosomiasis has occurred for more than 2000 years (Wei et al. 1981) and is still endemic in 32 out of the 71 counties with an estimated number of 260,000 patients. S. juponicum is a parasite not only of humans, but also of many other mammals and in particular of domestic livestock (Kumar et al. 1986). The overall infection rate of buffaloes was 5.85% for 1987 in Hubei Province (Wang and Jia 1989). Whereas the contamination of water bodies with human feces has now been greatly reduced by hygienic

measures, animal feces containing S. juponicum eggs are currently of prime importance for the continued transmission of this parasite to men. Control of schistosomiasis in China therefore substantially depends on success in the reduction of animal infections. Chemotherapy of domestic animals is a constituent of schistosomiasis control measures, but represents several drawbacks: chemotherapy with the highly effective drug pyquitone (praziquantel) is expensive, whereas other cheaper drugs (e.g., nithiocyamine) may have considerable side effects on the animals; the degree of reinfection after chemotherapy is unknown; and chemotherapy of ruminants requires injection of the drug. A veterinary vaccine may be an alternative to reduce the transmission of S. juponicum from animals to man. Veterinary vaccines based on y radiation-attenuated parasite larvae are available 100

0014-4894190 $3.00 Copyright 0 1990 by Academic Press, Inc. AU rights of reproduction in any form reserved.

VACCINATION

OF BUFFALOES

for large scale immunization of calves against Dictyocaulus viviparus (Poynter et al. 1970), dogs against Ancylostoma caninum (Miller 1978), and sheep against D. filaria (Sharma et al. 1988). The y-attenuated schistosome larvae have been used experimentally to immunize cattle against S. bovis in the Sudan (Bushara et al. 1978; Majid et al. 1980) and cattle as well as water buffaloes against S. japonicum in China (Hsu et al. 1983, 1984). The logistics of delivering the schistosome vaccine in these works present obvious difficulties: irradiated cercariae cannot be transported over long time periods, and transport of irradiated schistosomula requires continued deep freezing. Irradiation with ultraviolet (uv) light might be an alternative since equipment is portable and relatively cheap. Thus, cercariae could be attenuated in the field and might be used as a vaccine in an area endemic for schistosomiasis, but remote from central laboratory facilities. Immunization with uv-irradiated S. japonicum cercariae induces partial resistance against a challenge infection with normal cercariae in mice (Moloney et al. 1985a, b; Ruppel et al. 1990) and rats (Moloney et al. 1987). This paper evaluates the potential of uv-irradiated cercariae of S. japonicum to vaccinate water buffaloes (Bubalus bubalis) against S. japonicum infections. MATERIALS

AND METHODS

Oncomelania hupensis infected with S. japonicum were collected from the field in an endemic area of Hubei Province. Additional infected snails were purchased from the Schistosomiais Research Centre at Yao-Yang (Hunan Province). Cercariae were shed from about 150 snails illuminated overnight. If untreated, larvae produced with these techniques develop to adult worms in mice at a rate of about 50% (Ruppel ef al. 1990). Cercariae were irradiated in droplets of water on coverslips at 254 nm with an intensity of 400 pW I mm/cm2 for 60 set with a portable lowpressure mercury vapour uv lamp (type N 16; Konrad Benda, Laborgerate, 6908 Wiesloch, FRG). The energy output was controlled with a UV-CM equipment (same company) with a maximum sensitivity at 254

AGAINST

s.

101

japonicum

nm. The zero value of the UV-CM was calibrated for each irradiation period. Cercariae were used for immunization within 30 min after irradiation. This irradiation procedure reduces the viability of cercariae below O.l%, when determined in mice (A. Ruppel and Y.-E. Shi, unpublished results). Male water buffaloes aged between 6 and 9 months were purchased from farmers in the Wu-Jia-San village in an area of Hubei, which is not endemic for schistosomiasis. The absence of S. japonicum from the animals was controlled by examination of their feces in the miracidial hatching assay and of their sera in the indirect hemagglutination assay and circumoval precipitin test. All results were negative. Buffaloes were kept throughout the experiment at Dong-Shi-Hu Schistosomiasis Control Station, where no transmission of S. japonicum occurs. The buffaloes were immunized and infected on the back by applying the cercariae on coverslips to the shaven skin. Buffaloes were individually looked after by one person during the application of vaccine or challenge infections (30 mitt), such as to avoid undue movement and drying of the cercariae. Care was taken to apply each vaccination dose and challenge infection to a different area of the skin. Eight animals were immunized with 10,000 cercariae per dose at Days 0, 25, and 51. Six of these and six previously untreated buffaloes were challenged with 1000 untreated cercariae at Day 90. Two buffaloes were vaccinated, but not challenged, in order to check for worms developing from the uv-attenuated cercariae. Before the tirst immunization and at each occasion of subsequent immunizations and challenge infection, the rectal temperature was recorded, blood was obtained from all animals, and all were tested by the miracidial hatching assay for excretion of schistosome eggs. At the end of the experiment, the latter assay was repeated on 3 consecutive days. Results were only read qualitatively since counting of miracidia was difficult with large dung masses. Growth of the animals was followed by calculating their body weight according to the formula specified by the Institute of Animal Schistosomiasis, Shanghai: c’ x d

body weight (in kilograms) = 12,700 where c = circumference of thorax in centimeters determined at the height of the center of the scapula, and d = distance in centimeters from anterior superior edge of scapula to the sciatic end of the ilium. All buffaloes, except a nonvaccinated, but challenged one, which died of unknown reasons on Day 140, were killed and perfused between Days 144 and 145. Perfusion was done with the techniques developed at the Institute of Animal Schistosomiasis, Shanghai. Briefly, the portal vein was ligated close to

102

SHI ET AL.

the liver and distally incised; perfusion was done with water at tab pressure from the aorta. Subsequently, the portal vein was incised proximally of the ligature and reverse perfusion was done from the hepatic vein to wash out worms from the liver. Worms were collected in a nylon net, washed in physiological saline, and counted. Pieces of liver (5 to 10 g) were removed from identical locations of the right lobes. Schistosome eggs were counted after digestion for 2 hr at 37°C in 5% KOH. RESULTS

The body weight of vaccinated buffaloes increased continuously, although growth was slow during the later phases of the experiment. Previously untreated buffaloes lost body weight after the challenge (Table I). One of these animals died. The rate of the weight gain was initially slower in vaccinated as compared to untreated animals, but due to the weight loss of the latter group after the challenge, the overall increase in weight was ultimately similar for all animals (Fig. 1). The experiment was performed during the winter season, which might have entailed an exacerbation of the relatively poor health of the challenge control animals. However, the differences between both groups of animals were not statistically significant (P > 0.05) and further experiments should substantiate this data. The temperature of the animals was found to be normal and constant throughout the experiment: 38.3 ? 0.4”C and 38.1 + 0.4”C at the time of the first vaccination for, respectively, the prospective challenge control and vaccinated plus challenged animals; 37.8 k 1.2”C and 37.7 2 0.6”C at the time of perfusion. The worm burden of vaccinated and challenged buffaloes was reduced by 89% with respect to the worm burden of the challenge control group (Table I). Of the 30,000 uvattenuated cercariae used to vaccinate each buffaloe, only three worms per animal developed, representing a survival rate of 10m4. If these worms are substracted from those recovered from vaccinated and challenged buffaloes, a resistance of 92% re-

sulted in this experiment. The egg load in the liver was also greatly reduced in the vaccinated group vs control group (87%). The vaccination apparently did not affect the male:female ratio (1.07 + 0.09 in challenge control, 1.15 f 0.27 in vaccinated animals) nor the fecundity (expressed as eggs/ gram liverlschistosome pair) of the developing challenge-derived schistosomes. Excretion of S. juponicum eggs in the buffaloe feces could not be quantified in this experiment. In the miracidial hatching assay, only a few (two to six) miracidia per some 50 g of dung were detected; one vaccinated (number 7) and one vaccinated and challenged buffaloe (number 3) were, however, negative. DISCUSSION

We induced a strong resistance in water buffaloes against S. juponicum infections using uv-attenuated cercariae of this parasite. The success of the vaccination was evident in terms of three aspects: First, the level of resistance observed in the buffaloes (about 90%) was higher than the resistance obtained previously with the same techniques in most experimental rodent infections (ranging from about 30 to about 90% in different experiments; Moloney et al. 1985a, b, 1987; Ruppel et al. 1990). It also compares well with previous experimental immunizations of bovines with y-attenuated schistosomula: between 55 and 87% resistance against S. japonicum were induced in Holstein cattle (Hsii et al. 1983), while 65 to 72% and 74 to 76% resistance were induced in Chinese yellow cattle and water buffaloes, respectively (Hsii et ~1. 1984). Thus immunization with uvattenuated cercariae of S. juponicum appears to efficiently reduce the development of a challenge infection in buffaloes. Second, the vaccination with uvattenuated cercariae had a beneficial effect on the health status of the buffaloes following a challenge infection with normal cercariae. While nonvaccinated animals lost

1 6 9 12 13

Challenge Controlc,d

91.2 88.5 89.9 + 1.9

Beginning

136.8 109.2 114.8 89.8 127.8 115.7 k 18.1 104.9 117.3 161.2 170.1 105.4 191.2 141.7 2 37.1

115.8 104.1 109.9 k 8.2

Perfusion

S. japonicum:

63 30 55 76 44 10 3 9 4 6 2

12 5 9 4 6 2

2 1

Female

134 65 107 157 89 110.4 rt 36.3 22 8 18 8 12 4 12.0 2 6.8 89.1%

63 30 52 76 44

2 I

Pairs

Parasitological

4 2 3.0 k 1.4

Total

Number of worn&

71 35 52 81 45

2 1

Male

a Perfusion of buffaloes on Day 144. b Vaccination with doses of each 10,000 uv-irradiated cercariae at Days 0, 25, and 51. ’ Challenge with 1000 untreated cercariae on Day 90. ’ One animal died for unknown reasons 2 days before perfusion.

152.9 113.2 108.9 107.5 143.5 125.2 -+ 21.4 105.0 114.0 156.2 186.7 104.9 172.7 139.9 2 36.4

110.7 104.2 107.5 k 4.6

Challenge

Body weight (kg) at time of

116.7 79.3 93.9 73.5 99.1 Means 2 SD 92.5 + 17.0 Vaccinationb 2 87.4 Plus challenge’ 3 86.1 4 136.1 5 136.1 8 95.8 11 139.8 Means 2 SD 113.5 f 26.3 Reduction with respect to challenge control

7 10

Vaccinationb Control Means f SD

Treatment

Animal number

TABLE I Vaccination of Water Buffaloes with Ultraviolet-Irradiated Cercariae of

39 36 29 48 35 37.4 2 6.9 7 0 6 4 10 3 5.0 2 3.5 86.6%

0.57 1.20 0.56 0.63 0.80 0.75 + 0.27 0.70 0.00 0.67 1.00 1.67 1.50 0.81 f 0.60

4.0 2.0 3.0 + 1.4

Eggs/g/pair

Eggs in liver

8 2 5.0 + 4.3

&k

Data

104

SHI ET AL.

vaccines requires transport either of the bovines to the locality, where cercariae are irradiated, or of the frozen irradiated schistosomula to the bovines. Obviously, the latter situation would apply to a veterinary vaccine. Schistosomula of all major human schistosomes survive cryopreservation (James 1988) and radiation-attenuated cryopreserved schistosomula can be indeed successfully transported over long distances as shown for S. mansoni (James et al. 1986). However, in a situation where “we Time slier first immtizabn (months) should regard schistosomiasis japonica as a FIG. 1. Influence of vaccination with uv-attenuated disease of bovines which may infect man cercariae of S. japonicum on the growth of water buf- instead of a disease of man which may infaloes. Buffaloe calves were vaccinated at times 0, 1, fect bovines” (Hsti et al. 1984), more pracand 2 months with doses of 10,000 uv-irradiated certicable and cheaper ways of vaccine delivcariae (n = 8, W) or remained untreated (n = 6, 0). ery are desirable. We propose such a posAll animals except two vaccinated ones were challenged with 1000 normal cercariae at 3.5 months. The sibility in this report. experiment was terminated at 5 months. Body weight Vaccination with a high number of attenwas calculated as described under Materials and Methuated larvae might present a health risk for ods. Absolute values (see Table I) were set at 100% at host. An obvious one the time of first immunization. The standard devia- the schistosome would originate from worms, which might tions are indicated. develop from the irradiated vaccine, and this argument holds particularly true for body weight, the vaccinated ones continvaccination of humans (von Lichtenberg ued to gain weight. This was probably due 1985). The survival in bovines of vaccineto the reduction of the tissue egg load (87% derived worms was not tested in previous in the liver) of vaccinated buffaloes. In pre- reports concerning bovines, while in our vious field trials with y-attenuated schisto- two vaccinated, but not challenged, buffasomula of S. japonicum, vaccinated and loes a survival rate of 1O-4 was found for challenged cattle and buffaloes had, respec- the 30,000 irradiated cercariae applied per This low number of vaccinetively, 70 and 81% fewer eggs in the liver as animal. derived worms is expected from the rate of compared to challenge control animals; inactivation of cercariae by uv-light, when they also gained weight, whereas unvaccinated controls lost weight (Hsti et al. 1984). determined in mice (Ruppel et al. 1990). It The uv-attenuated vaccine therefore ap- would not appear, however, to be a serious pears to be just as beneficial as a y-irradrawback of the immunization procedure diated one. under field conditions, where the worm Third, the delivery of the uv-attenuated burden resulting from natural exposure durvaccine in this report was inexpensive (cost ing a few months to cercariae might be 20 of lamp plus uv meter about 1100 DM, no times as high in a lightly endemic area and maintenance, expected life time 2,500 hr), 250 times higher in an area of heavy endepractical (weight of equipment less than 2 micity (Hsti et al. 1984). kg, largest dimension 35 cm, 220 V plug), During the vaccination period, the and easily applicable under simple field growth rate of animals was slowed down. conditions (we used it in a farmer’s house). This suggests a possible adverse effect of In contrast, the delivery of the y-attenuated the vaccine on the buffaloes’ health, al-

VACCINATION

OF BUFFALOES

though this was entirely compensated for by the vaccine-mediated protection against the challenge-induced weight loss. The influence of the vaccine on weight gain may depend on the amount of attenuated cercariae used; whether fewer cercariae may provide protection in the absence of adverse effects will be studied. Acute side effects of the vaccine were not detected as judged from the body temperature of the buffaloes. However, we cannot yet rule out possible transitory fever periods after vaccination. The available data on vaccination of bovines against S. japonicum with attenuated larval schistosomes (Hsti ef al. 1983, 1984 and this report) is consistent with earlier results on vaccination of zebu calves with y-irradiated schistosomula or cercariae of S. bovis (Bushara et al. 1978, Majid et al. 1980). In these experiments, vaccinated and challenged animals had greatly reduced worm burdens and tissue egg loads, excreted fewer eggs, and gained weight in comparison to challenged, but not vaccinated animals. The present immunization protocol with uv-attenuated cercariae of S. japonicum might be improved in the future, e.g., in order to possibly reduce the amount of cercariae and the number of vaccinations required. The time period during which animals will maintain a high level of vaccineinduced resistance is also an important factor to be determined. The immune mechanisms of, and schistosome developmental targets for, the immune response of buffaloes remain completely unknown. The debate on the mechanisms of vaccineinduced resistance against S. mansoni continues (McLaren 1989; Wilson and Coulson 1989) and may be relevant for S. japonicum as well. Our current studies of the humoral immune response of buffaloes might shed some light on this issue. Buffaloes appear to have an innate level of resistance greater than cattle in that only between 6% (Hsu et al. 1984) and 11% (this report) of the infect-

AGAINST

s.

105

japOniCUm

ing normal cercariae develop in buffaloes to adult worms, while well above 50% do so in cattle (Hsti et al. 1983,1984). Evidence also suggests that buffaloes may cure infections spontaneously (Ho 1963; Zhang et al. 1989). This innate resistance may contribute to the efficiency of a uv vaccine for calf buffaloes. “If schistosomiasis (japonica) in bovines is not controlled, this disease will never be controlled in humans” (Hsu et al. 1984). We would certainly endorse this statement for the Hubei Province. We hope that the present approach for vaccination will contribute to reducing the transmission of S. japonicum from domestic animals to man. ACKNOWLEDGMENTS We thank Mr. S. S. Li (Dong-Shi-Hu Schistosomiasis Control Station, Hubei Province) for excellent cooperation and the staff of the Institute of Animal Schistosomiasis (The Chinese Academy of Agricultural Sciences, Shanghai) for kindly teaching us the perfusion techniques of water buffaloes. This work received tinancial support from the Stiftung Volkswagenwerk (I/ 63469), the Forschungsschwerpunkt HD19 of BadenWilrttemberg, and from the Hubei Scientific Research Committee. REFERENCES BUSHARA, H. O., HUSSEIN, M. F., SAAD, A. M., TAYLOR, M. G., DARGIE, J. D., MARSHALL, T. F. DE C., AND NELSON, G. S. 1978. Immunization of calves against Schisrosoma bovis using irradiated cercariae or schistosomula of S. bovis. Parasitology 77, 303-311. CHEN, M. G. 1988. Schistosomiasis control in China. In “XIIth International Congress for Tropical Medicine and Malaria” (P. A. Kager and A. M. Polderman, Ed.), pp. 243-244. Excerpta Medica, Amsterdarn/New York/Oxford. CHENG, T. H. 1971. Schistosomiasis in mainland China. American Journal of Tropical Medicine and Hygiene

20, 26-53.

Hsii, S. Y. L., HsO, H. F., Xu, S. T., SHI, F. H., HE, Y. X., CLARKE, W. R., AND JOHNSON, S. C. 1983. Vaccination against bovine schistosomiasis japonica with highly X-irradiated schistosomula. American Journal giene 32, 367-370.

of Tropical

Medicine

and

Hy-

Hsii, S. Y. L., Xu, S. T., HE, Y. X., SHI, F. H., SHEN, W., Hsii, H. F., OSBORNE, J. W., AND CLARKE, W. R. 1984. Vaccination of bovines

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against schistosomiasis japonica with highly irradiated schistosomula in China. American Journal of Tropical Medicine and Hygiene 33, 891-898. JAMES, E. R. 1988. Cryopreservation of parasites. In “Parasitology in Focus” (H. Mehlhom, Ed.), pp. 684-701. Springer-Verlag, Berlin/Heidelberg/New York. JAMES, E. R., OTIENO, M., HARRISON, R., DOBINSON, A. R., MONOREI, J., AND ELSE, J. G. 1986. Partial protection of baboons against Schistosoma mansoni using radiation-attenuated cryopreserved schistosomula. Transactions ofthe Royal Society of Tropical Medicine and Hygiene 80, 378-84. KUMAR, V., AND DE BURBURE, G. 1986. Schistosomes of animals and man in Asia. Helminthological Abstracts 55, 469-480. VON LICHTENBERG, F. 1985. Conference on contended issues of immunity to schistosomes. American Journal of Tropical Medicine and Hygiene 34, 78-85. MAJID, A. A., BUSHARA, H. O., SAAD, A. M., HusSEIN, M. F., TAYLOR, M. G., DARGIE, J. D., MARSHALL, T. F. C., AND NELSON, G. S. 1980. Observations on cattle schistosomiasis in the Sudan, a study in comparative medicine. III. Field testing of an irradiated Schistosoma bovis vaccine. American Journal of Tropical Medicine and Hygiene 29,452455. MAO, S. P., AND SHAO, B. R. 1982. Schistosomiasis control in the People’s Republic of China. American Journal of Tropical Medicine and Hygiene 31, 9299. MCLAREN, D. J. 1989. Will the real target of immunity to schistosomiasis please stand up. Parasitology Today 5, 279-282. MILLER, T. A. 1978. Industrial development and field use of the canine hookworm vaccine. Advances in Parasitology 16, 333-342. MOLONEY, N. A., BICKLE, Q. D., AND WEBBE, G. 1985a. The induction of specific immunity against Schistosoma japonicum by exposure of mice to ultra violet attenuated cercariae. Parasitology 90, 3 13323.

MOLONEY, N. A., GARCIA, E. G., AND WEBBE, G. 1985b. The strain specificity of vaccination with ultra violet attenuated cercariae of the Chinese strain of Schistosoma japonicum. Transactions of the Royal Society of Tropical Medicine and Hygiene 19, 245-247. MOLONEY, N. A., WEBBE, G., AND HINCHCLIFFE, P. 1987. The induction of species-specific immunity against Schistosoma japonicum by exposure of rats to ultra-violet attenuated cercariae. Parasitology 94, 49-54. POYNTER, D., PEACOCK, R., AND MENEAR, H. C. 1970. The prevention and treatment of husk. Veterinary Record 86, 148-160. RUPPEL, A., SHI, Y. E., AND MOLONEY, N. A. 1990. Schistosoma mansoni and S. japonicum: Comparisons of levels of ultraviolet irradiation for vaccination of mice with cercariae. Parasitology, in press. SHARMA, R. L., BHAT, T. K., AND DHAR, D. N. 1988. Control of sheep lungworm in India. Parasitology Today 4, 33-36. WANG, 2. L., AND JIA, Y. D. 1989. Prevalence and control of schistosomiasis in China (in Chinese language). Chinese Journal of Schistosomiasis Control 1, 6-8. WEI, D. X., YANG, W. Y., HUANG, S. Q., Lu, Y. F., Su, T. C., MA, J. H., Hu, W. X., AND XIE, N. F. 1981. Parasitological investigation on the ancient corpse of the Western Han Dynasty unearthed from tomb No. 168 on Phoenix hill in Jiangling County. Acta Academiae Medicinae Wuhan 1981, 16-23. WILSON, R. A., AND COULSON, P. S. 1989. Lungphase immunity to schistosomes: A new perspective on an old problem. Parasitology Today 5, 274-278. ZHANG, C. F., Luo, X. F., AND LING, S. Y. 1989. A study on the relationship between antibody level and survival of adult worms of Schistosoma japonicum in experimentally infected water buffaloes (in Chinese language). Chinese Journal of Schistosomiasis Control 1, 48-50. Received 30 October 1989; accepted with revision 28 January 1990

Schistosoma japonicum: an ultraviolet-attenuated cercarial vaccine applicable in the field for water buffaloes.

Water buffaloes were vaccinated three times with 10,000 Schistosoma japonicum cercariae irradiated with ultraviolet (uv) light at a dose of 400 microW...
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