Department of Helminthology, Faculty of Tropical Medicine, Mahidol University 420/6 Rajavithi Road, Bangkok 10400, Thailand

Эта статья опубликована в сборнике по материалы первой Международной юбилейной конференции «Актуальные проблемы инфектологии и паразитологии» , посвященной 110-летию со дня открытия проф. К.Н.Виноградовым сибирской двуустки у человека (2-5 апреля 2001, г. Томск)

Посмотреть содержание сборника

Скачать сборник целиком

Хромосомы и C-районы в хромосомах у Opisthorchis viverrini

Резюме
Хромосомы O. viverrini изучали после высушивания,  методикой С-зондирования. Диплоидный набор хромосом составил 2n=12, гаплоидный набор n=6. Гаплоидный набор включал в себя: один крупный метацентрик, один средний метацентрик, один маленький метацентрик, один маленький субметацентрик или субтелоцентрик, один небольшой субтелоцентрик или акроцентрик и один маленький акроцентрик. Относительные размеры хромосом составляли: 32,02+2,52; 23,28+1,98; 15,24+3,40; 13,39+3,11; 10,18+1,56 и 5,82+0,59%, соответственно. После С-зондирования в двух небольших хромосомах был выявлен конститутивный гетерохроматин.

Abstract

Chromosome of Opisthorchis viverrini was observed by air-drying and C-banding techniques. The chromosome number was 2n=12 and n=6 consisting of one large-sized metacentric, one medium-sized metacentric, one small-sized metacentric, one small-sized submetacentric or subtelocentric. one small-sized subtelocentric or acrocentric and one small-sized acrocentric. The relative lengths of the chromosomes were 32.02 ± 2.52. 23.28 ± 1,98, 15.24 ± 3.40. 13.39 ±3.11. 10.18 ± 1.56 and 5.82 ± 0.59 % respectively. After C-banding treatment, two of the small-sized chromosomes showed a remarkable constitutive hetcrochroma-tin.

INTRODUCTION
The liver fluke, Opisthorchis viverrini has been recognized as the only species of harmful parasite being associated definitively with human cancer (cholangiocarcinoma) in Thailand. Many aspects of this parasite have been studied, while its chro¬mosomes have not yet been investigated. A small number of reports have been available in the litera¬ture and there have been a few studies on the chromo¬some of genus Opisthorchis. Jha (1975) reported that the chromosome of 0. felineus was 2n= 16, n=8 and 0. i;et-minus was 2n=18. n= 9. Cho (1978) also reported that the chromosome number of Clononhis sinensis (Opisthorchis sinensis) was 2n= 14. n=7. This paper aimed to check and demon¬strate the chromosome of 0. viverrini using con¬ventional Giernsa staining as well as C-banding methods.

MATERIALS AND METHODS
Specimen preparation
Cyprinoid fishes, known to be the intermedi¬ate host of 0. viverrini were collected from a large swamp in the Chonabot district. Khon Kaen Province. northeast Thailand. The metacercariae of 0. viverrini were obtained from these fishes. After oral inoculation of metacercariae, 40-50 days later. adult specimens were recovered from the livers of golden hamsters.
Chromosome preparation
  The Terasaki (1977) air-drying technique was modified and used lor chromosome preparation. Live specimens were put into a colchicine-salt solution (0.005% w/v colchicine in 0.859r sodium chloride solution) for three hours at 37oC. One ovary and two testes per adult fluke were taken out with a micropin under a dissecting microscope. Then each of the ovaries and testes was put on a glass slide with a few drops of 0.6% sodium citrate solution, and was broken with a micropin under a dissecting microscope. Germ cells were spread out on the glass slide and were kept at room temperature for thirty minutes. Then these glass slides were put into a moisture box which contained Carnoy solution (methyl alcohol 1: acetic acid 1). After thirty min¬utes, Carnoy solution was put onto the glass slides with a pipette and five minutes later the solution was shed and blown dry. All slides were stained with 4% Giemsa solution pH 6.8 for thirty minutes at room temperature before conventional investiga¬tion.
C-banding technique
   Chromosome banding of 0. viverrini was per¬formed by the Hirai (1985) method with a little modification. Dried chromosome preparations were left in a 37°C incubator for at least 1 week and then treated directly with 5% barium hydroxide solution for 10 minutes, followed by 2SSC (Standard saline-citrate) solution for 60 minutes. Both treatments were set at 60^C. Slides were stained with 4% Giemsa solution pH 6.8 for 90 minutes at room tempera¬ture. All preparations were then used for microscopic observation.

RESULTS
In the metaphase figures of germ cells of 0. viverrini, the chromosome number was 2n=12, n=6 and divided into three groups (large, medium and small) according to their size. Based on chromosome nomenclature recommended by Levan et al (1964), they consisted of one large-sized metacentric, one me¬dium-sized metacentric, one small-sized metacentric, one small-sized submetacentric or subtelocentric, one small-sized subtelocentric or acrocentric and one small-sized acrocentric. On the basis of 27 metaphase figures, the relative length of each chro¬mosome was calculated. After C-banding treatment, two of the small-sized chromosomes of 0. viverrini showed a remarkable con¬stitutive heterochromatin.

DISCUSSION
Jha (1975) studied the chromo¬some numbers of liver flukes from sectioned materials in India and found that the chromosome numbers of 0. felineus and 0. germinus were 2n = 16, n = 8 and 2n =18, n = 9 respectively. Cho et al (1978) also reported that the chromosome number of C. sinensis in Japan was 2n =14, n =7. Ilyinskikh (1982) found that the chromosome number of 0. felineus in Siberia was 2n =14, n =7. The present study revealed that the chro¬mosome number of 0. viverrini in Thailand was 2n =12, n = 6, which was remarkably different from 0. felineus, 0. germinus and C. sinensis.
   C sinensis, 0. felineus and 0. viverrini have been known to be the causative agents of liver fluke infection in a number of countries. 0. viverrini, has been found to be the only species of liver fluke infection in Thailand (Maleewong et al, 1992; Radomyos et al, 1994; 1998) while C. sinensis is prevalent in China, Taiwan, Korea, Japan and North Vietnam (Choi, 1984; Cross, 1984; Chen et al, 1997). 0. felineus used to be prevalent in Central and Eastern Europe and Siberia, especially in Germany and Poland (Beaver et al, 1984); currently this fluke is still endemic in Germany, Siberia and the Ukraine (Hinz, 1991; Hering-Hagenbeck et al, 1996;Loktieva,1997). 0. viverrini and 0. felineus are closely related species in terms of morphology of the adult flukes except that they are a little different in the shape of the testes and vitellaria, while both species are markedly different from C. sinensis (Beaver et al, 1984). The morphological characteristics of eggs and metacercariae of the three species are similar and difficult to differentiate from each other. Therefore, it is considered that the significant differences of chromosome numbers among 0. viverrini, 0. felineus, and C. sinensis may reflect genetic distinctions of the respective parasite groups, geo¬graphic distances and their biological differences. The finding may be used for identification and taxonomic purposes, especially in distinguishing between 0. felineus and 0. viverrini.
   C-banding pattern has been used to study sev¬eral aspects of closely related parasites and primates (Hirai et al, 1985; Zhao et al, 1989; Tan and Li 1990; Pieczarka et al. 1998). The present study re¬vealed that C-band formation in the chromosome of 0. viverrini was only seen in two of the small-sized chromosomes. Up to the present time, there have been no reports concerning this aspect in other species of Opisthorchis and Clonorchis. This paper is the first report of demonstrated C-banding in the chromomsome of 0. viverrini. Further study should emphasize phylogeny among the three species of liver flukes from different localities using biochemical and molecular analysis.

REFERENCES
Beaver PC, Jung RC, Cupp EW. Clinical Parasitology. 9th ed. Philadelphia: Lea and Febiger; 1984.
Chen S, Chen S, Wu F, et al. Epidemiological survey on clonorchiasis sinensis in Yangxin County of Hubei Province of PR China. Southeast Asian J Trop Med Public Health 1997; 28 (suppl 1): 51-3.
Cho H. Studies on the chromosomes of parasitic helm¬inths (1). Chromosomes in meiocytes, spermiogen-esis and fertilization observed by means of a squash method in Clonorchis sinensis (Trematoda : Opisthorchiidae). Jpn J Parasitol 1978; 27: 399-410.
Choi DW. Clonorchis sinensis: life cycle, intermediate hosts, transmission to man and geographical distribution in Korea. Arzneimittelforschung 1984; 34: 1145-51.
Cross JH. Changing patterns of some trematode infections in Asia. Arzneimittelforschung 1984; 34: 1224-6.
Hering-Hagenbeck S, Schuster R. A focus of opisthorchiidosis in Germany. Appi Parasitol 1996; 37:260-5.
Hinz E. Current status of food-bome parasitic zoonoses in West Germany. Southeast Asian J Trop Med Public Health 1991; 22: 78-84.
Hirai H, Sakaguchi Y, Habe S, Imai HT. C-banding analysis of six species of lung flukes, Paragonimus spp (Trematoda: Platyhelminthes), from Japan and Ko¬rea. Z Parasitenkd 1985; 71: 617-29.
Ilyinskikh NN, Bocharov EF, Ilyinskikh IN. Infection mutagenesis, Nauka, Novosibirsk, Russia. 1982:184.
Jha AG. Cytogenetics, evolution and systematics of digenea.(Trematoda : Platyhelminths), Egypt J Genet Cytol 1975; 4: 201-33.
Levan A, Fredca K, Sandberg A. Nomenclature for centromeric position on chromosomes. Hereditas 1964;
52:201-20.
Loktieva IM. An opisthorchiasis epidemic in Ukraine. Lik Sprava 1997;3:146-9.
Maleewong W, Intapan P, Wongwajana P, et al. Preva¬lence and intensity of Opisthorchis viverrini in rural community near the Mekong river on the Thai-Laos border in northeast Thailand. J MedAssoc Thai 1992; 75:231-35.
Pieczarka JC, Nagamachi CY, Muniz JA, Barros RM, Mattevi MS. Analysis of constitutive heterochroma-tin of Aotus (Cebidae, Primates) by restriction en¬zyme and fluorochrome bands. Chromosome Res 1998;6:77-83.
Radomyos P, Radomyos B, Tungtrongchitr A. Multi-in¬fection with helminths in adults from northeast Thai¬land as determined by post-treatment fecal exami¬nation of adult worms. Trop Med Parasitol 1994;
45:133-5.
Radomyos B, Wongsaroj T, Wilairatana P, et al. Opisthorchiasis and intestinal fluke infections in northern Thailand. Southeast Asian J Trop Med Public Health 1998;29:123-7.
Tan Q, Li D. Study on chromosome c-banding in Paragonimus westermani: comparison of the diploid and triploid types. Chung Kuo Chi Sheng Chung Hsueh Yu Chi Sheng Chung Ping Tsa Chih 1990; 8: 35-7.
Terasaki K. Studies on Chromosomes of the lung flukes in Japan. Jpn J Parasitol 1977; 26: 222-9.
Zhao JB, Huang SY, Zhao SY. Comparative study on chro¬mosome C-banding in diploid and triploid types of Paragonimus westermani. Chung Kuo Chi Sheng Chung Hsueh Yu Chi Sheng Chung Ping Tsa Chih 1989;7:103-4.