Gnat.Hostoma Spinigeruoij
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ผลธองรังสีแกมมาจากโคบอลท้ -60 ต่อตัวออนระยะตดต่อออง'พยาธิตัวจีด 'Gnat.hostoma spinigeruoiJ " ประเสริฐ เสดสุบรรท1 * ปีณไทวุพเ หิรักชุเะร'าสิธ•าดา * ล'มจิตร ภู่บำเนก! ยุทธ•พง ประปีฯลทธิสืกด วิ?ต โรจ'นกิตติคุทเ * หารผ ดคำย้อย *ภา«วิธฯบ่าราสิตหนอน'พยาธิ ค[นะเ วขค่า?)ดรเขตร้อน มาทวิทยาลัยมหดล **กองวิทยาค่าส์ดร์ขิ'วภา'พ สำนักงาน'พลงงานปรมาณูเหื่อสันติ บทศัดย่อ จากการสืกษา'พบว่า ตัวอ่อนระยะติดต่อชอง'พยาธิตัวจด (Gnathostoma spinigerum) มีความด้านทานลูงดอรังสีแกมม่า โดยที่มันล่ามารถมี?วิตรอดและก่อให้เกิลการ ติดเสือ•พยาธินในหนทดลอง (mice) ได้ ถงแม้จะได้รับปริมาณรังสีล'งถง 7 กิโลเกรย ตัวอ่อน ของ'พยาธิคัวเดระยะติดต่อที่ได้รับการฉายรังสีขนาด 0, 0.3, 0.6, 1.0, 1.5, 2.0, 3.0, 5.0, และ 7.0 กิโลเกรย์ จะมิอัตราการก่อให ้ เกิดการติด เขี1อ'พยาธิในหนูทดลองเป็น 72%, 78%, 64°/, 54°/, 28%, 42%, 26%, 14% และ 5% ตามลำดับ ตัวอ่อน'พยาธิที่ได้รับการฉาย รังสี ขนาด 8 และ 10 กิโลเกรย์ ไม่ลฯมาร.",ก่อให้เกิดการติดเจอ'พยาธิในหนูทดลองได้ ต้ว อ่อน'พยาธิจะเรีมมีการเดสีอนใหวที่เปีองข้ฯลงกว่าปกติเมื่อได้รับปริมาณรังสี ดั้งแคปีนาด 1.5 กิโลเกรย์ และเคลอนไหวเสืองขาลงเรอย ๆ รวมทั้งมีดวามลามารถในการก่อให้เกิดการติดเสือ 'พยาธิในหนูลดลงเมีอได้รับปริมา™รังสีที่มากขน ตัวอ่อนหยาธิที่ได้รับรังสีขนาด 10 กิโลเกรย์ ใ?ง เป็นธนาดลูงสุดในการทดลองนยังล่ามารถมีสืวิต แต่ไม่อาจก่อให้เกิดการติดเสือ'พยาธิในหนูทดลอง ได้ จากผลการทดลอง'พบว่าปริมาณรังสีอนาด 8 กิโลเกรย เป็นขนาดตํ่าสุดที่ลามารกยับยั้งการก่อ ให้เกิดการติดเสือ'พยาธิปีนิดนในหนูทดลอง นอกจากนยังได้ทำการทดลองฉายรังสีคัวอ่อน'พยาธิที่ ผล'มลงในรมปีก โดย'ใช้บริมาท!รังสือนาด 8 กิโลเกรย์ เหอทดสอบยนย้นทงปริมา[นรังสีที่ทอว่า เป็นรนาดที่ยับยั้งการก่อให้เกิดการติดเสือ'พยาธิในหนทดลยิง 66 Effects of Cobalt-60 Gamma Radiation on Gnathostoma spinigerum larvae Abstract Gnathostoma spini.gerirm infective larvae were found to be very resistant to gamma ray. The larvae were still viable, motile and capable to infect white mice with no observable abnormality in both internal and external structures and their sizes after exposure to 0 - 7.0 KGy of gamma ray. At doses of 0, 0.3, 0.6, 1.0, 1.5, 2.0, 3.ช, 5.0 and 7.0 KGy, the infective rates were 72%, 78%, 64%, 54%, 28%, 42%, 26%, 14% and 5% respectively. The infective rates of the irradiated larvae exposed to 8 KGy and 10 KGy of irradiation in mice became zero. The reduction of motility and infectivity was first observed after exposure of larvae to 1.5 KGy of gamma ray. It was also found that motility and infectivity decreased in relationship to the higher doses of gamma ray. At 10.0 KGy of irradiation which was the highest dose used in this experiment could not devitalize the larvae but inhibit infectivity. Irradiation of the larvae mixed in food prepared from fishes at 8 KGy was proved to inhibit the infectivity. On the stu<iy, the dose of 8.0 KGy was suggested to be the minimum effective dose that inhibited the infectivity of the infective stage. To confirm the effective dose, the mixture of the infective larvae and Somfuk, a local food, was treated by 8 KGy and these irradiated larvae precisely lost their infectivity in mice. 67 Introduction There are 22 species of Gnathostoma that have been reported in the world but 12 species are valid.5 Only four species are known causing diseases in man namely G. spinigerum,5 G. hispidum e, G. do loresi 9 and G. nipponicum.1 Five species of Gnathostoma are reported in man and animals in Thailand namely G. spinigerum,5 G. hispidum, G. doloresi,6 G. vietnamicum 5and G. malaysiae 7and only G. spinigerum is proven causing human gnathostomiasis. The distribution of G. spinigerum is also in U.S.A., บ.S.S.R., Northern Rhodesia, Australia, Palestine, Japan, India, China, Philippines, Vietnam, Malaysia, Bangladesh and Myanma5. In Thailand, up date 47 species of vertebrates including fresh water fishes, brackish water fishes, reptiles, rodents, amphibians and avians are able to serve as the natural second intermeditae hosts of G. spinigerum and some vertebrates act as experimental second intermediate hosts or paratenic hosts such as pig or chicken5' Approximately, over 500 suspected cases of gnathostomiasis came to attend the gnathostomiasis clinic at chulalongkorn hospital and Tropical Medicine hospital per year. Fatal cases are occasionally reported. Gnly one worm can cause death in man and no drug of choice can kill this worm13 . In many parts of Thailand, people consume various products of fermented or pickled raw pork or fish. The most popular one is called Moo-nham or Somfuk which made of pork or fish respectively. This study will investigate the usefulness or effects of Cobalt-60 gamma radiation on G. spinigerum larvae. If the radiation is proved to be effective on G. spinigerum larvae, it will be an important role to prevent or control of gnathostomiasis. 68 Materials and Methods Phase 1 Preparation of G. spinigerum larvae Eggs of G. spinigerurn were collected from feces of infected cats by means of brine floatation technique. Eggs were washed several times with dechlorinated water to remove debris and the remaining brine by centrifuge . These eggs were then placed in a petri-dish containing small amount of boiled water and left at the room temperature. After hatching and developing, the first stage larvae were put in container rearing numerous fresh water cyclops. The examination for the infected cyclops with early third stage larvae was done on day 8 after post-infection . Phase '?. Irradiation of larvae In vivo to find Minimum Effective Dose (MED> Thirty early third stage larvae were fed on each laboratory mouse. These infected mice were divided into two groups, group I used as control and another as experiment. The control and the experimental groups were then subdivided into eleven groups. Each group, except control, was exposed to gamma ray from Co-60 at various doses of 0, 0.3, 0.6, 1.0, 1.5, 2.0, 3.0, 5.0, 7.0, 8 and 10 KGy respectively. The radiation doses were confirmed by using optic chromic, nylon thin film (FWAT-60-00) and Red Perspex dose meter which were attached on the body of mice for each dose of irradiation. The irradiation treatment was done in the laboratory of the Atomic Energy for Peace, Bangkok. After the treatment, all mice were dissected to recover cysts or larvae in mice tiss.e at the Faculty laboratory. The larvae removed from the irradiated mice were tested for viability, morphological deformity or change, infectivity and growth. The infectivity was tested in a group of ten mice, each mouse was fed with 5 irradiated larvae. The control group was fed with non-irradiated larvae done by the same process. The animals were then kept for three weeks before being sacrified. Then each group of mice both control and experiment was killed and carefully removed and 69 examined skin and internal organs under stereomicroscope. All tissues were chopped into small pieces and digested by 1% acid pepsin solution at 37 °c for 2-4 hours for presenting the larvae which were determined the following respects of viability, morphological change, growth and percentage of infectivity. Phase 3 The infectivit.y of larvae treated with minimum effective dose Advanced-third stage larvae of G. spinigerum obtained from experimental mice were mixed in Somfuk, the local - fermented food product prepared by raw freshwater fishes. The irradiation of Somfuk was determined at the dose of 8 KGy which had suggested to be the minimun effective dose inhibiting development of infective larvae in Phase 2. After removing the irradiated larvae, each of ten mice was fed on 5 larvae by stomach tube and the control was done with ร of non-irradiated larvae by the some process. Mice were sacrified for searching larvae after three weeks of post infection. Criteria for determining the viability of G. spinigerum larvae Observation of larvae which were transferred into physiological saline from 5 minutes until overnight are determined as follows; 1. Larvae that show movement when stimulated with chemicals light, heat, warm water or touching with insect pin are considered alive, whereas the ones that have no respond are considered dead. 2. Dead larvae show structural changes such as destruction of cuticle, swollen body, presence of fine granules or air bubbles in the internal organs and showing sign of degeneration. Results 1. Morphology of the irradiated larvae The larvae which were exposed to 0-10 KGy showed no abnormality in their morphology and size when compared to the 70 non-Irradiated larvae in control group (Table I). 2. Motility of the irradiated larvae Irradiated larvae from mice exposed to gamma ray at 0-10 KGy showed motility. The motility would reduce inversely to the increasing doses of gamma ray. The motility of larvae at doses 7.0, 8.0, 10 KGy would not be observed immediately or at a period of 1-3 hours after exposure to the radiation but motility of many larvae could be observed in the solution at the room temperature after they were left overnight. 3. Recovery of irradiated larvae from mice Mice infected with irradiated G. spinigerum larvae at doses of 0-7 KGy of gamma ray showed the negative rate for larvae with the percentage of 30%, 20*, 40%, 50% and 75% at the doses of 1.5, 2.0, 3.0, 5.0 and 7.0 KGy respectively. At the doses of 8.0 and 10.0 KGy, the mice were totally negative from the larvae (Table II). After larvae were exposed to gamma ray at each experimental doses, these larvae were fed into mice, the infectivity or larva recovery rates in mice were 72%, 78%, 64%, 54%, 28%, 42%, 26%, 14%, 5%, 0% and 0% respectively (Table III). 4. Infectivity of irradiated larvae The 8 KGy - treated larvae removed from irradiated Somfuk revealed normal morphology comparing to the control and still moved in physiological saline. After gamma ray treatment, these infective larvae could not penetrate stomach wall of mice while the non-irradiated larvae penetrated into tissue of the control (Table IV). Discussion Food-Borne parasitic Zoonosis (FBPZ) may impact on agriculture, industry, economic and Public Health. Gamma irradiation is a very useful tool for controling FBPZ. Dubey et. al. 19863 71 reported that the minimum effective dose (MED) of gamma ray for controling Toxoplasma gondii was 0.5 KGy whereas Chancun et.