FULL PAPER Parasitology

The Effect of Heating against Oocysts

Takashi FUJINO1), Toshihiro MATSUI1), Fumie KOBAYASHI1), Kosuke HARUKI1), Yukiko YOSHINO2), Junko KAJIMA3) and Moriyasu TSUJI1)

1)Division of Tropical Diseases and Parasitology, Department of Infectious Diseases, Kyorin University School of Medicine, 6–20–2 Shinkawa, Mitaka-shi, Tokyo 181–8611, 2)Kyorin University School of Health Sciences, 476 Miyashitamachi, Hachioji-shi, Tokyo 192– 8508 and 3)Research and Study Department, Japan Association of Parasite Control, 1–2 Sadohara-cho, Ichigaya, Shinjuku-ku, Tokyo 162–0842, Japan

(Received 21 August 2001/Accepted 14 November 2001)

ABSTRACT. The effect of heat treatment was examined against oocysts of , Cryptosporidium muris and chicken Cryptosporidium sp. isolated in Japan. The oocysts of these species were exposed at 50, 55, 60 and 70°C for 5, 15, 30 and 60 sec in water bath, respectively. To determine the infectivity of heated oocysts, the mice and chickens were inoculated with the treated oocysts and the oocyst output in the feces after inoculation was examined. In C. parvum and chicken Cryptosporidium sp., the oocysts were not detected from mice or chickens which were received oocysts heated at 55°C for 30 sec, 60°C for 15 sec and 70°C for 5 sec. In C. muris, the oocysts were not detected from mice which were received oocysts heated at 55°C for 15 sec, 60°C for 15 sec and 70°C for 5 sec. Consequently, it was clarified that the infectivity of Cryptosporidium oocysts to mice and chickens was lost by heating at 55°C for 30 sec, 60°C for 15 sec and 70°C for 5 sec. KEY WORDS: chicken Cryptosporidium, Cryptosporidium muris, Cryptosporidium parvum, infectivity of heated oocyst. J. Vet. Med. Sci. 64(3): 199–200, 2002

Cryptosporidiosis is a zoonotic diarrheal disease caused week-old chickens (White Leghorn). The mice and chick- by the oocysts of Cryptosporidium parvum [1]. Moreover, ens were confirmed to be free of natural coccidial infections the waterborne outbreaks with Cryptosporidium parvum by fecal examinations prior to experiments and were housed occurred in many countries [2, 7, 8, 12]. However, a safe- separately as groups in wire-bottom cages and raised under guard against Cryptosporidium oocysts has not yet been the -free environment. clarified. It is generally known that the coccidian oocysts Experimental design: This experiments were designed are sensitive to heat exposure though they are resistant to according to the procedure of Ito et al. [6]. Briefly, the dis- disinfectants. Therefore, the effects of heat treatment were tilled water (30 ml) in the glass centrifuge tubes were incu- examined against oocysts of Cryptosporidium parvum, bated in water bath at 50, 55, 60 and 70°C. One ml distilled Cryptosporidium muris and chicken Cryptosporidium sp. water containing 2.6 × 107 oocysts were dropped into the isolated in Japan. each tube. The oocyst suspension was also dropped into the tubes at 25°C as a non-treated control. The each sample was MATERIALS AND METHODS incubated for 5, 15, 30 or 60 sec. After heat treatment, the samples were added immediately into 80 ml of cold distilled Oocysts: C. parvum (strain SC1) used in this study was water (4°C). The samples were centrifuged at 2,000 rpm for first isolated from the feces of naturally infected Siberian 7 min. Then the sediment was resuspended in 3 ml of dis- chipmunks as companion animals purchased from commer- tilled water. The number of oocysts in the final suspension cial sources [10]. C. muris (strain RN66) isolated from the was counted using a plankton counting plate [9]. Five SCID house rat was supplied by Dr. Iseki [4], Osaka City Univer- mice, 5 ICR mice and 3 chickens each were inoculated with sity Medical School. Cryptosporidium sp. from the chicken 2.5 × 106 C. parvum or C. muris oocysts or 3.2 × 106 chicken was obtained from Dr. Itakura [5], Faculty of Veterinary Cryptosporidium sp. oocysts from each final suspension and Medicine, Hokkaido University. These species were sub- oocyst output was examined by sugar flotation method. jected to passage in SCID mice, ICR mice or chickens to multiply oocysts, respectively. The oocysts were collected RESULTS from feces of the mice or chickens, and mixed with distilled water. The oocysts were purified by sugar centrifugal flota- In case of C. parvum and chicken Cryptosporidium sp., tion method (specific gravity of sugar, 1.153). The superna- the mice or chickens received the oocysts which were tants were washed with distilled water by centrifugation treated at 55°C for 30 sec or more, 60°C for 15 sec or more (2,000 rpm for 7 min). The oocysts were stored at 4°C in 2% and 70°C for 5 sec or more did not shed any oocysts (Tables potassium dichromate solution and were used within 1 1, 3). month. In case of C. muris, the mice received the oocysts which Experimental animals: Animals used in this study were 5- were treated at 55°C for 15 sec or more, 60°C for 15 sec or week-old mice (SCID), 3-week-old mice (SPF, ICR) and 2- more and 70°C for 5 sec or more did not shed any oocysts 200 T. FUJINO ET AL.

° Table 1. Oocyst production in mice inoculated with heat treated oocysts was lost after exposure at 70 C for 2 min that the oocysts of Cryptosporidium parvum Toxoplasma oocysts had similar characteristics to eimerian oocysts in the sensitivity to heating. In the present experi- Duration of Temperature of exposure (°C) ments, we examined the sensitivity to heating by the same exposure (sec) 50 55 60 70 procedure described by Ito et al. [6], and found that the 5+++–infectivity of three species of Cryptosporidium oocysts was 15 + + – – lost by heating at 70°C for 5 sec. Therefore, it was clarified 30 + – – – that the cryptosporidian oocysts had higher sensitivity to 60 + – – – heat exposure than Toxoplasma and Eimeria oocysts. +: Oocyst positive. –: Oocyst negative. From these results, it is considered that the hot-water heaters are useful for disinfection of contaminated cow- Table 2. Oocyst production in mice inoculated with heat sheds, sheepfolds and chicken houses with coccidian para- treated oocysts of Cryptosporidium muris sites if they are appropriately equipped. Duration of Temperature of exposure (°C) exposure (sec) 50 55 60 70 REFERENCES

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