Carcinogenic Human Liver Fluke : Current Status of Opisthorchis viverrini Metacercariae in Nakhon Ratchasima , Thailand

Opisthorchis viverrini, fish-borne trematode infection causes a serious public-health problem in Southeast Asia mainly Thailand, Cambodia, and Laos PDR. In Thailand, opisthorchiasis is a major public health problem in the Northeastern and Northern region, and it is estimated that 5.5 million people are infected with the causative parasite (Sripa et al., 2010). Human have been infected by ingesting undercooked fish containing infective metacercariae, this is very common in the northeastern and northern region particularly in rural areas (Sadun, 1955; Sithithaworn et al., 1997; Jongsuksuntigul & Imsomboon, 2003). The infection is associated with a number of hepatobiliary diseases, including cholangitis, obstructive jaundice, hepatomegaly, cholecystitis and cholelithiasis (Harinasuta et al., 1984). The experimental and epidemiological evidences strongly indicate that the liver fluke infection in the etiology of cholangiocarcinoma (CCA); the bile duct cancer (Thamavit et al., 1978; IARC, 1994). Multi-factorial etiology of CCA, mechanical


Introduction
Opisthorchis viverrini, fish-borne trematode infection causes a serious public-health problem in Southeast Asia mainly Thailand, Cambodia, and Laos PDR.In Thailand, opisthorchiasis is a major public health problem in the Northeastern and Northern region, and it is estimated that 5.5 million people are infected with the causative parasite (Sripa et al., 2010).Human have been infected by ingesting undercooked fish containing infective metacercariae, this is very common in the northeastern and northern region particularly in rural areas (Sadun, 1955;Sithithaworn et al., 1997;Jongsuksuntigul & Imsomboon, 2003).
Many pathological features in the hamster model reveal evidence of such mechanism, e.g.periportal infiltration of lymphocytes and macrophages.Acute damage may be induced by parasite factors, but the progressive changes are consistent with immunopathologic mechanisms (Pairojkul et al., 1991).During liver fluke infection, inflammation, periductal fibrosis, and proliferative responses, including epithelial hyperplasia, goblet cell metaplasia, and adenomatous hyperplasia, may represent predisposing lesions that enhance susceptibility of DNA to carcinogens (Kim, 1984;Flavell & Flavell, 1986).Several N-nitroso compounds and their precursors occur at low levels in fermented food, such as preserved mud fish paste, pla ra, a condiment that is a ubiquitous component of the cuisine of Northeastern Thailand and Laos (Sripa et al., 2007).Some authors hypothesised that N-nitroso compounds are a primary carcinogen leading to CCA in humans in Northeastern Thailand (Migasena et al., 1980;Srisraluang & Boriboon, 1988).Jinawath et al showed the selective up-regulation of genes involved in xenobiotic metabolism in Thai patients with CCA, implying that these genes may be involved in the detoxification of possible carcinogens, such as nitrosamines (Jinawath et al., 2006).In hamster experimentally infected with O viverrini, CCA was induced by exposure to sub-carcinogenic doses of nitrosamine (Thamavit et al., 1978).
Liver fluke infection can induce the endogenous nitrosation both in humans and experimental animals.Infected hamster showed nitric oxide synthase (NOS) expression by immune effector cells in the inflamed areas and increased endogenous nitrosation of thiazolidine-4carboxylic acid (thioproline) (Srianujata et al., 1987).In human studies, infected cases showed a higher endogenous nitrosation than uninfected cases (Srivatanakul et al., 1991;Satarug et al., 1998).Infected cases had increased levels of plasma and urinary nitrate and nitrite compared to the uninfected cases (Srianujata et al., 1987).Praziquantel treatment and co-administration of ascorbic acid with proline showed enhanced immune responses to O viverrini and an increase of endogenous nitrosation (Oshima et al., 1994).Thus the aforementioned findings clearly demonstrate both exogenous and endogenous nitrosation may lead to DNA alkylation and deamination in predisposed and inflamed tissues (Miwa et al., 1987;Nguyen et al., 1992).
In Thailand, fish-borne trematode infections have been commonly found in the Northeastern and Northern regions of Thailand (Harinsuta & Vajrasthira, 1960;Wykoff et al., 1965;Vichasri et al., 1982).However, lack of data about the metacercariae; O. viverrini infection in Nakhon Ratchasima province, Thailand which is the largest province on the northeastern plateau and acts as a gateway to other provinces in the Northeastern region of Thailand.Therefore, we have constructed the cross-sectional research to survey and determined the metacercariae from the natural cyprenoid fish.This research data could be used to localize the risk areas to prevent the infection.

Materials and Methods
The study protocol was approved by Suranaree University Biotechnological Review Committee.Cyprinoid fish, the common intermediate host for O. viveriini, were collected from 32 districts of Nakhon Ratchasima province, Thailand.It is 259 kilometres from Bangkok and has an territory area of around 20,494 square kilometres, making it the biggest province in Thailand.Cyprinoid fish were collected at 32 water reservoirs; Mueang Nakhon Ratchasima, Khon Buri, Soeng Sang, Khong, Ban Lueam, Chakkarat, Chok Chai, Dan Khun Thot, Non Thai, Non Sung, Kham Sakaesaeng, Bua Yai, Prathai, Pak Thong Chai, Phimai, Huai Thalaeng, Chum Phuang, Sung Noen, Kham Thale So, Sikhio, Pak Chong, Nong Bun Mak, Kaeng Sanam Nang, Non Daeng, Wang Nam Khiao, Thepharak, Mueang Yang, Phra Thong Kham, Lam Thamenchai, Bua Lai, Sida, Chaloem Phra Kiat districts from February 2010 to February 2011 (Figure 1).These fish were collected by fisherman using nets (Figure 2), dead fish were kept in labeled plastic bags and transported in an ice box to the laboratory at the Parasitic Disease Research Unit, department of Pathology, Institute of Medicine, Suranaree University of Technology, Thailand, within a day after collection.The fish were separated and categorized as species according to their morphological characteristics.For each species, fish samples were photographed and kept in a preserved state with labels, for further identification to species; the rest were used in the study.After thorough washing under tap water, and then inspected for metacercariae using the standard Pepsin-HCl digestion method (Sithithaworn et al., 1997;Srisawangwong et al., 1997).Fishes were digested using an acid pepsin solution (1 ml conc.hydrochloric acid, 1 g pepsin, 99 ml 0.85% sodium chloride solution for 1 1/2 hours at 37ºC.The digested material was then rinsed with 0.85% sodium chloride solution and examined for metacercariae.Encysted metacercariae were dissected from the cyprinoid fish under a stereomicroscope and placed in NSS.They were mounted individually on a glass slide, covered with a coverslip, and examined under amicroscope.Occasionally they were excysted by gentle pressing with the cover slip for more detailed examination in an extended condition.The metacercariae were identified with the aid of morphological criteria such as the size of cysts, folding body displaying vigorous movement within the cyst, prominent and clearly visible oral and ventral suckers (Vajrasthira et al., 1960;Scholz et al., 1991;Kaewkes, 2003).Finally, the numbers of cyprinoid fish were infected with O. viverrini metacercariae were recorded.The O. viverrini juveniles were removed, counted, fixed in 4% formalin, and stained with Borax's carmine or hematoxylin, dehydrated in an alcohol series, and mounted with Permount.The species were identified after Yamaguti (1958Yamaguti ( : 1963)), Pearson (1964), Kliks & Tantachamrun (1974), Pearson & Ow-Yang (1982), Radomyos et al. (1990) and Kaewviyudth (1998).
The prevalence of O. viverrini metacercariae in fish was analyzed descriptively using Excel data sheet.The prevalence of infection was expressed as the percentage of the number of metacercariae-positive fish/total number of fish examined.
The prevalence varied from 6.49% to 19.16% depending on fish species, with the mean prevalence of 12.34% in the total of 640 cyprinoid fishes.Among those, Cyclocheilichthys armatus and C. repasson showed relatively high (>15%) prevalence.The prevalence of other three cyprinoid species were <10%.Encysted metacercariae were oval in shape and covered with a thin single layer of cyst wall.The metacercariae had large oral and ventral suckers with a tubular excretory bladder at the posterior end, and were moving actively in the cyst (Figure 3).The excysted metacercariae; juvenile worms were lancet-shaped, about 0.24 mm long, and were morphologically identified as O. viverini.The morphological identification of metacercariae and the excysted worms was confirmed further by genetic analyses.
More than half (78.13%) of the natural reservoir sites were selected showed that cyprinoid fish were infected with O. viverrini metacercariae.The infection rates of O. viverrini in 32 districts for the year-round survey are    shown in Table 3 and Figure 4.In Kham Thale So and Sida districts were found the highest infection rate, 8 of 20 fish in 5 species harbored metacercariae of O. viverrini while in 6 districts mainly Nong Bun Mak, Wang Nam Khiao, Huai Thalaeng, Pak Chong, Soeng Sang Khong, and Pak Thong Chai were not infected.

Discussion
Opisthorchis viverrini, fish-borne trematode infection causes a serious public-health problem in Southeast Asia mainly Thailand, Cambodia, and Laos PDR.In Thailand, opisthorchiasis is a major public health problem in the Northeastern and Northern region, and it is estimated that 5.5 million people are infected with the causative parasite (Sadun, 1955;Jongsuksuntigul & Imsomboon, 2003;Sripa et al., 2010).The infection is associated with a number of hepatobiliary diseases, including cholangitis, obstructive jaundice, hepatomegaly, cholecystitis and cholelithiasis (Harinasuta et al., 1984).The experimental and epidemiological evidences strongly indicate the liver fluke infection in the etiology of cholangiocarcinoma (CCA); the bile duct cancer (Thamavit et al., 1978;IARC, 1994).Multi-factorial etiology of CCA, mechanical damage, parasite secretions, and immunopathology may enhance cholangiocarcinogenesis (Flavell, 1981;Sripa et a., 2007).Human have been infected O. viverrini by ingesting undercooked fish containing infective metacercariae, this is very common in the northeastern and northern regions particularly in rural areas.Three types of preparations contain uncooked, small and medium-sized, fish: koi pla, eaten soon after preparation; moderately fermented pla som; stored for few days to weeks; pla ra extensively fermented, highly salted fish, stored for at least 2-3 months (Sithithaworn et al., 1997).O. viverrini infection is commonly found in the northern and northeastern regions of Thailand.Several species of cyprinoid fish have been reported as the second intermediate hosts of O. viviverrini (Harinsuta & Vajrasthira, 1960;Wykoff et al., 1965;Vichasri et al., 1982).In our study, 5 species of cyprinoid fish were found infected with O. viverrini metacercariae.Among the metacercariae, O viverrini had the highest number found throughout the year-round survey.O. viverrini was predominantly found in several species of fish namely Cyclocheilichthys armatus, C. repasson, Puntioplites proctzysron, Hampala macrolepitota, and Hampala dispar, respectively.These figures showed the Similarity to the other research such as Khemphavanh et al (2009) concerning frequent infection in these cyprinoid fish species.Our study is the first report of O viverrini infected fish in Nakhon Ratchasima province Thailand that is different to other known reports.Nithiuthai et al. (2002) reported that no fish infected with metacercariae were found.However, they had surveyed in a small scale and specific sample with short period of time in the Lum Cha Muak stream, Nakhon Ratchasima province; the study was conducted during May 2000.Many factors can affect the prevalence of metacercariae infection in fish, including the setting, season, type, and number of parasites and fish species.Generally, the highest metacercarial infection rate in fish is found during late rainy season in October.The low prevalence in their study, in May, might therefore be lower than the actual peak prevalence in the late rainy season (Vichasri et al., 1982, Sithithaworn et al., 1997).Our study is set up to cover the areas of province and also select the samples throughout the year-round survey.Moreover, the Bureau of Epidemiology, Department of Disease Control, Ministry of Public Health, Thailand have been reported that Nakhon Ratchasima is still the site to find the patients infected with O. viverrini (2010: 2011).Humans are not the only source of O. viverrini.Fish-eating animals can also be involved in their life cycle (Faust & Nishigori, 1926;Pearson 1964;Ditrich et al., 1990).Food-borne trematodes can be assessed by the prevalence of infections in several animal life cycles.
The distribution of food-borne trematodes depends on many factors, such as the relationship between host, parasite, environment, and traditional food habits (WHO, 1995).Kham Thale So and Sida district showed the highest prevalence of metacercarial infection in fish.It is possible that this area contained highest number of patients and it's also a high-risk area in Nakhon Ratchasima Province for O. viverrini infection.In addition, Thai traditional dishes, such as 'Pla-som' and 'Koy-Pla', are the most common sources of infection in people.These raw-fish dishes cannot induce degeneration of the contaminated metacercariae a short period of time (Sukontason al., 1998;Wiwanitkit et al., 2003), and there is a tendency that the prevalence of human infection will increase because of eating raw fish.We have no data concerning prevalence of O. viverrini infection in 32 districts, Nakhon Ratchasima province.Further study, to compare and determine the relationship, Should be done for screen the current status, their knowledge, attitude, and behavior of people to O. viverrini.
The result of this study was a practical understanding of fish-borne trematode infections in Nakhon Ratchasima Province, Thailand.Infection rates and distribution of O. viverrini metacercarial infection among certain species of cyprinoid fish were demonstrated in the Sikhio and Chaloem Phra Kiat reservoires.Further research studies in endemic areas of Nakhon Ratchasima Province, Thailand, should consider interventions using health-education and sanitation-improvement approaches in the liverfluke control program, to change eating and defecation habits, and thereby reduce the occurrence of O. viverrini infection.
In conclusion, our study showed the update prevalence of O. viverrini metacercaria in cyprinoid fish in Nakhon Ratchasima Province.This may be caused by unsanitary rural latrines.Eggs and larvae of O. viverrini can contaminate reservoirs by running water and precipitation.O. viverrini can infect humans who eat undercooked freshwater fish.The data from our study can help rural people who use this contaminated water to prevent parasitic infections in the future.

Figure 1 .
Figure 1.Collection of Cyprinoid Fish Collected from Natural Reservoirs of Various 32 Districts, Nakhon Ratchasima Province Thailand, by local Fishermans using nets.