Clonorchiasis is an infectious disease caused by the liver fluke Clonorchis sinensis, which often accompanies serious symptoms such as hepatic dysfunction and liver cancer. High prevalence of C. sinensis infection has been increasingly reported in many parts of Vietnam. C. sinensis uses freshwater snails of the genus Bithynia as its primary hosts and freshwater fish species as its secondary hosts, and human infection occurs when metacercariae in flesh or on skin of freshwater fish are ingested by human hosts. The disease transmission is influenced by various cultural and socioeconomic factors such as distribution of intermediate host snails, contamination of aquatic environment by human excreta, culturing methods of freshwater fish, a habit of raw fish consumption, and host-parasite relationship. It is thus important to develop an area-specific control approach by understanding the existing transmission cycle of C. sinensis in each local setting and identifying the point of intervention where the transmission cycle can be effectively cut off.

Since freshwater fish is the major transmission media of C. sinensis infection to human, expansion of freshwater aquaculture has been implicated as a potential cause of high clonorchiasis frequency in east and Southeast Asia. However, due to the lack of substantive information to confirm the association between the recent rapid development of freshwater aquaculture sector and the prevalence of C. sinensis, no control strategies have been established targeting at freshwater fish production. As a result, the national control program of foodborne trematode infections in Vietnam has only focused on mass drug treatment and health education.

The present study aimed at elucidating the association between the prevalence of C. sinensis infection and freshwater fish consumption/production in Vietnam, and thereby recommending a control strategy to produce hygienically safe freshwater fish while evading associated health risks rather than to change people' tradition and cultural behavior by external intervention. The study focused on the Red River delta of northern Vietnam where C. sinensis infection is reportedly the most prevalent in the country.

In Chapter 2, the association between the prevalence and the intensity of C. sinensis infection by age, gender and a habit of eating raw fish was assessed in the area of high-prevalence and low-prevalence of C. sinensis infection in northern Vietnam.

Stool examination and questionnaire survey were conducted in Kim Son district of Ninh Binh province where consumption of raw freshwater fish had been widely known (the first study) and Luong Son district of Hoa Binh province where such consumption had not been reported at the time of the survey (the second study), targeting at 1,155 and 155 villagers respectively.

In the first study, 648 out of 1,155 individuals (56%) reported the habit of eating raw fish and the prevalence of infection was 26% (301). In the second study, 35 out of 155 (23%) individuals reported the same habit and the infection prevalence was 1.3% (2). The extremely low number of positive cases made odds ratio analysis impossible in the second study. In the first study, however, the infection prevalence was 3-fold higher in males than in females, and the habit of eating raw fish increased the risk of C. sinensis infection by 53-fold. The strong association of raw fish consumption with the prevalence and the intensity of C. sinensis infection were thus confirmed in this chapter.

Freshwater fish is one of the most important protein sources for Vietnamese citizens, especially in non-coastal regions. Freshwater fish production in the Red River delta has been steadily growing since 1990 in terms of both production and area. Chapter 3 attempted to investigate the association between the production systems of freshwater fish and the prevalence of C. sinensis infection in the Red River delta, Vietnam. The types of freshwater aquaculture in the Red River delta were found to be 3-fold: small-scale aquaculture, large-scale aquaculture and rice-cum-fish culture. The most dominant in the delta was small-scale aquaculture, accounting for approximately half of the water surface used for freshwater aquaculture, followed by large-scale aquaculture. This study revealed a tendency that the larger the area of water surface used for aquaculture in provincial land was, the higher the reported provincial prevalence of C. sinensis infection was. The infection prevalence was particularly high where small-scale aquaculture was extensive. In large-scale aquaculture and rice-cum-fish culture systems, commercial pellet feed is commonly used. In the meantime, small-scale aquaculture was further sub-divided into a feed-based system and an fertilizer-based system, and in the latter case, animal manure and human excreta are reused to fertilize fish ponds in place of commercial feed. This led author to speculate that the direct use of animal manure and human excreta in fertilizer-based systems, which accounted for a substantial portion of small-scale aquaculture, caused harboring of various pathogens including C. sinensis in these fish ponds, infected the farmed fish in the ponds with C. sinensis, and resultantly led to the high prevalence of C. sinensis infection in the provinces where small-scale aquaculture was prevalent.

In Chapter 4, the prevalence of C. sinensis infections in relation to engagement in aquaculture and more specifically, the use of animal manure and/or human excreta in aquaculture were assessed in order to confirm the public health concern on this waste reuse in freshwater aquaculture. Stool examination and questionnaire survey were carried out in Kim Son district of Ninh Binh province where the first survey of Chapter 2 was performed and all the participants of the study who were found to be infected with C. sinensis received treatment with praziquantel. The re-infection rate of the study population was 9.2% (19 positive cases out of 206). While engagement in agriculture did not contribute to increased risk of C. sinensis infection, aquaculturists were found to show 2.9-fold higher infection risk than non-aquaculturists (p<0.001). Contrary to author's speculation that the use of animal manure and/or human excreta in aquaculture led to the high prevalence of C. sinensis in the provinces where small-scale aquaculture was prevalent, the use of neither of animal manure nor human excreta increased the infection risk (OR 0.4, p=0.21 for animal manure; OR 0.2, p=0.06 for human excreta). Besides, composting of animal manure and/or human excreta did not reduce the infection risk (p=0.48 by Fisher's exact test for animal manure; p=0.08 by Fisher's exact test for human excreta). This indicated that farmed fish were equally parasitized by C. sinensis regardless of the use of animal manure or human excreta. It was suggested that there were other factors that contributed to parasitization of freshwater fish by C. sinensis in fish farming areas.

For freshwater fish to be parasitized by C. sinensis, excreta of C. sinensis-infected humans or animals need to enter the water body where the fish inhabits, and snail hosts also need to inhabit in the same water body. Chapter 5 aimed at comparing the fecal contamination level of water environment of different aquaculture systems, namely excreta-based systems (using animal manure) and feed-based systems (using commercial feed), in the Red River delta of Vietnam. Escherichia coli (E. coli) was used as an indicator of fecal contamination. Concentrations of E. coli in fish pond water and feedwater (irrigation water) as well as those on the skin surface of the farmed fish (Nile Tilapia Oreochromis niloticus) were enumerated. E. coli counts on the skin surface of tilapia were strongly correlated with those in pond water (R2=0.90, 9<0.05). The E. coli counts of both the fish pond water and on skin surface of the farmed fish were significantly higher in fertilizer-based systems than in feed-based systems. This is presumably due to the direct use of fresh animal manure in fertilizer-based systems. According to multiple regression analysis, the number of pigs kept per m2 pond area was found to be an important determinant of the fecal contamination level not only of pond water but also of fish skin. The E. coli level of feedwater did not significantly differ between the two systems since both fertilizer-based and feed-based ponds co-existed in the study area and the same irrigation water was fed into the both systems. In the study area, not only the E.coli counts in the pond water of both systems but also those of the local irrigation canal exceeded the microbiological quality standards of wastewater-fed aquaculture recommended by WHO (1.0 x 103 E.coli per 100ml water sample) and that of water for fisheries designated by the Japanese government (1.0 x 103 E.coli per 100ml water sample). This implied that the fecal contamination of the canal water occurred due to the fact that both excreta-based and feed-based ponds co-existed in the area, from which pond water is equally discharged back to the canal two to three times a month.

Chapter 6 provided an overall discussion of the results presented in Chapters 2 to 5. On the basis of the findings, the potential transmission mechanism of C. sinensis in aquaculture-active areas was speculated as follows: firstly, the reuse of pig manure to fertilizer fish ponds in fertilizer-based systems contaminated both pond water and farmed fish in the system; secondly, discharge from the fertilizer-based systems into the local irrigation canal contaminated the irrigation water; thirdly, this irrigation water was fed into fish ponds of feed-based systems then contaminated both pond water and farmed fish in the system. Accordingly, it was conclude that inappropriate management of the fertilizer-based aquaculture systems was causing parasitization of farmed fish by C. sinensis in both systems.

Conventional control strategies of C. sinensis infection are comprised of the following two approaches: 1) drug treatment of patients with praziquantel for eliminating human host reservoir; and 2) health education for a promotion of cooked fish consumption for preventing infection. It is, however, widely acknowledged that mass treatment of patients combined with health education alone is not sufficient to eradicate the infection, as people's cultural behavior and food habits cannot be changed easily. Establishment of a clonorchiasis control strategy targeting at freshwater fish production is therefore expected.

The livestock-integrated aquaculture systems in Vietnam are known to be highly cost-effective, environmental-friendly as it effectively recycles agricultural wastes, and has contributed to poverty reduction of poor farmers in Vietnam. In view of the rapid growth of aquaculture sector in Vietnam, this chapter recommended strategic measures to produce hygienically-safe freshwater fish by reducing the health impacts associated with this system, such as pretreatment of feedwater before intake or that of pond water prior to discharge, and eradication of snail hosts in both aquaculture ponds and irrigation canals by use of molluscicide or introduction of their natural predators, and to ultimately promote it as a model for sustainable fish production.

シナ肝吸虫症はシナ肝吸虫が肝臓に寄生することによってひきおこされる感染症で,肝機能不全や肝癌などの重篤な疾病をしばしば引き起こす.シナ肝吸虫は淡水性巻貝のマメタニシを第一中間宿主とし,淡水魚を第二中間宿主とする.人間への感染は,淡水魚に寄生したメタセルカリアを摂取することによって起きる.疾病の伝播は,さまざまな文化的・社会経済的な要因による影響を受ける.したがって,地域ごとに感染サイクルを理解し,効果的な感染防止策を立てる必要がある.そのような中にあって,東アジア・東南アジアでは,淡水魚の養殖が伝播に関係しているとの説がある.ベトナムでも,シナ肝吸虫症感染の増加が報告されている.しかし,最近のシナ肝吸虫症の増加と,水産養殖の普及との関係を裏付ける実証的な研究はない.そのため,ベトナムでは駆虫薬の投与が唯一の吸虫感染症対策となっている.本研究の目的は,ベトナムのシナ肝吸虫症と養殖の普及の関係を実証的に確かめ,衛生的な淡水魚の生産法を提言することによって,人々の食文化を変更することなく,シナ肝吸虫症の伝播をたちきることにある.

序章に引き続いて第2章では,シナ肝吸虫の感染率が高い地域と低い地域において,性別,学歴,年齢,生活習慣等とシナ肝吸虫への感染の関係を調べた.その結果,男性は女性よりも感染率が高く,淡水魚を生食する習慣を持つ者は持たないものに比べて,53倍の感染確率を持つことが明らかとなり,シナ肝吸虫症の感染が主として淡水魚の生食によって起きていることが確認された.

第3章では,淡水魚の養殖方法と寄生虫感染症の関係を調査した.統計によると,ベトナムの紅河デルタ地帯には大規模養殖・小規模養殖・稲田養殖の3タイプの淡水魚の養殖がある.このうち,小規模養殖は全養殖面積のおよそ半分を占める.感染頻度が高い地域は,小規模養殖が盛んな地域であった.ベトナムの小規模養殖のほとんどは,家畜し尿や人糞などの施肥によって魚を生産する複合養殖の形態である.一方,大規模養殖と稲田養殖は給餌養殖であり,し尿の施肥をほとんど行わない.そのことから,複合養殖におけるし尿の施肥が寄生虫の伝播に大きく関係しているものと推測した.

第4章では,前述の推測とシナ肝吸虫症防除に関する知識普及の効果を確かめるために,8年前に,シナ肝吸虫症防除のプロジェクトが行われた地域において,駆虫薬の投与を受けた者に対して検便検査を行った.その結果,9.2%の再感染者がいることがわかった.感染者の多くは,淡水魚の生食が主たる感染原因であるという知識を持っていたが,多くの者が淡水魚の生食を続けていた.また,淡水魚を生食する者の80%が自家生産した養殖魚を食べており,そのうち32%が家畜し尿を,31%が人糞を施肥に利用していた.しかし,し尿の施肥養殖をおこなう者の間の感染確率は,施肥養殖をおこなう者に比べて低い値であった.このことから,施肥養殖,給餌養殖にかかわらず,地域全体の魚がシナ肝吸虫に汚染されていることを示しているものと推測した.

第5章では,地域全体がシナ肝吸虫の汚染されるメカニズムを解明するため,大腸菌を指標として,養殖方法と糞便汚染の関係を調べた.その結果,施肥養殖では,豚のし尿や人糞をコンポスト化することなく直接に,養殖池に施肥していた.養殖池の水中および養殖魚の体表の大腸菌数は,施肥養魚池で有意に高かったが,給餌養殖の飼育水および池の給水中の大腸菌数も我が国およびWHOの養魚用水基準よりも高い値であった.その原因として,調査地域では,養魚用水の給水路と排水路が分離されておらず,養魚用水は灌漑水路から取水され,灌漑水路に直接排出されているためだと考えられた.

第6章では,シナ肝吸虫症の感染サイクルを断ち切る方法について考察した.調査の結果,知識の普及によって,人々の食習慣を変えることは難しく,駆虫薬の投与後も再感染の可能性が高いことがわかった.したがって,養殖技術の改善によって,地域全体として感染サイクルを断ち切ることが必要である.確実な対策は,養魚用水の給排水を分離することであるが,これをベトナムの現状で実施することは予算的に困難である.現状においては,灌漑水路の水をいったん処理池で処理して給水するか,排水時に処理をして水路に排水することが現実的な方法と考えられた.また,し尿を直接施肥せずに,コンポスト化して利用するよう教育指導を行うことも有効な対策と考えられた.

以上,本研究は,寄生虫感染という公衆衛生上の問題を,養魚技術の改良によって解決しようとしたものであり,発展途上国における公衆衛生上の問題を解決するうえで示唆に富んでいる.また,水産学の研究に新しい展開をもたらす可能背もある.よって審査委員一同は本研究を博士(農学)の学位論文として価値あるものと認めた.