Research and Reviews ill Parasitology. 57 (3-4): 145-218 (1997) Published by A.P.E. © 1997 Asociaci6n de Parasit61ogos Espafioles (A.P.E.) Printed in Barcelona. Spain

HUMAN LIVER FLUKES: A REVIEW

S. MAS-COMA & M.D. BARGUES Departamento de Parasitologia. Facultad de Farmacia, Universidad de Valencia, Av. vicent Andres Estelles sin, 46100 Burjassot - Valencia, Spain

Received 21 Apri11997; accepted 25 June 1997 REFERENCE:MAS-COMA(S.) & BARGUES(M. D.). 1997.- Human liver flukes: a review. Research and Reviews in Parasitology, 57 (3-4): 145-218. SUMMARY:Human diseases caused by are reviewed. The present knowledge on the following 12 digenean species belonging to the families Opisthorchiidae, Fasciolidae and is analyzed: , Opisthorchis feline us, O. viverrini, Fasciola hepa- tica, F. gigantica, , D. hospes, Eurytrema pancreaticum, Amphimerus pseudofelineus, A. noverca, Pseudamphistomum truncatuin, and Metorchis conjunctus. For each species the following aspects of the parasite and the disease they cause are reviewed: morphology, location and definitive hosts. reports in humans, geographical distribution. life cycle. first intermediate hosts, second intermediate hosts if any, epi- demiology. pathology. symptomatology and clinical manifestations. diagnosis, treatment, and prevention and control.

KEY WORDS: Human diseases, Clonorchis sinensis, , O. viverrini, , F. gigantica,Dicrocoelium dendriticum, D. hospes, Eurytrema pancreaticum, Amphimerus pseudofelineus, A. noverca, Pseudamphistomum truncatum, Metorchis conjunctus, review.

CONTENTS

Introduction 147 Clonorchis sinensis 148 Morphology .. 148 Location and definitive hosts. 148 Reports in humans .. 149 Geographical distribution 149 Life cycle. 150 First intermediate hosts 151 Second intermediate hosts 152 Epidemiology 152 Pathology, symptomatology and clinical manifestations 153 Diagnosis 154 Treatment. 155 Prevention and control 155 156 Morphology. 156 Location and definitive hosts 157 Reports in humans 157 Geographical distribution 158 Life cycle 158 First intermediate hosts. 159 Second intermediate hosts 159 Epidemiology 160 Pathology, symptomatology and clinical manifestations. 162 Diagnosis. 164 Treatment 165 Prevention and control. 165 Opisthorchis felineus 166 Morphology 166 Location and definitive hosts 166 Reports in humans .. 167 Geographical distribution. 168 Life cycle 168 First intermediate hosts 168 Second intermediate hosts 168 Epidemiology 168 Pathology, symptomatology and clinical manifestations 170 Diagnosis 170 Treatment 171 Prevention and control 171 Fasciola hepatica 171 Morphology .. 171 Location and definitive hosts 172 146 S. MAs-CoMA & M.D. BARGUES

Reports in humans. 172 Geographical distribution 173 Life cycle 175 First intermediate hosts . 175 Epidemiology. 176 Pathology, symptomatology and clinical manifestations. 177 Diagnosis. 179 Treatment. 181 Prevention and control. 182 . 182 Morphology. 182 Location and definitive hosts. 183 Reports in humans. 183 Geographical distribution. 184 Life cycle. 184 First intermediate hosts. 184 Epidemiology. 185 Pathology, symptomatology and clinical manifestations . 185 Diagnosis. 186 Treatment. 186 Prevention and control. 186 Dicrocoelium dendriticum . 186 Morphology. 186 Location and definitive hosts. 187 Reports in humans. 187 Geographical distribution. 188 Life cycle. 188 First intermediate hosts . 189 Second intermediate hosts. 189 Epidemiology. 189 Pathology, symptomatology and clinical manifestations . 190 Diagnosis. 191 Treatment. 191 Prevention and control. 192 Dicrocoelium hospes 192 Morphology . 192 Location and definitive hosts. 192 Reports in humans. 192 Geographical distribution. 193 Life cycle. 193 First intermediate hosts. 193 Second intermediate hosts. 193 Epidemiology. 194 Pathology, symptomatology and clinical manifestations . 194 Diagnosis. 194 Treatment. 194 Prevention and control. 194 Eurytrema pancreaticum . 194 Morphology. 194 Location and definitive hosts. 194 Reports in humans. 195 Geographical distribution. 195 Life cycle. 195 First intermediate hosts. 195 Second intermediate hosts. 196 Epidemiology. 196 Pathology, symptomatology and clinical manifestations . 196 Diagnosis. 196 Treatment. 196 Prevention and control. 196 Amphimerus pseudofelineus . 196 Amphimerus noverca . 197 Pseudamphistomum truncatum . 197 Metorchis conjunctus . 197 References. 198 Introduction. 198 Clonorchis sinensis . 198 Human liver flukes: a review 147

Opisthorchis viverrini. 200 Opisthorchis [elineus .. 203 Fasciola hepatica . 205 Fasciola gigantica. 210 Dicrocoelium dendriticum. 212 Dicrocoelium hospes .. 215 Eurytrema pancreaticum .. 216 Amphimerus pseudofelineus . 217 Amphimerus noverca .. 217 Pseudamphisiomum truncatum .. 217 Metorchis conjunctus. 217

INTRODUCTION cal importance owing to the large number of people in- fected (the estimated number of people infected can be Among digenean parasites affecting the liver of hu- counted in millions in each case), allowing us to really man beings, the following 12 species belonging to three speak of human endemic areas (see RIM et al., 1994). trematode families are involved: The species D. dendriticum, F. gigantica and D. hospes, A) Opisthorchiidae: Clonorchis sinensis (Cobbold, 1875) in this order, constitute an intermediate group according Looss, 1907; Opisthorchis felineus (Ri volta, 1884) to their importance in humans, human cases being relati- Blanchard, 1895; Opisthorchis viverrini (Poirier, 1886) vely numerous but usually isolated (the estimated num- Stiles et Hassall, 1896; Amphimerus noverca (Braun, ber of people infected can be counted in hundreds in 1902) Barker, 191I; Amphimerus pseudofelineus each case). Finally, E. pancreaticum, P. truncatum, A. (Ward, 1901) Barker, 1911 (= Opisthorchis guayaqui- pseudofelineus, A. noverca and M. conjunctus have been lensis Rodrfguez G6mez et Montalvan, 1949); Metor- reported in humans only very sporadically (less than ten chis conjunctus (Cobbold, 1860) Looss, 1899; and published cases for each parasite), although recent stu- Pseudamphistomum truncatum (Rudolphi, 1819) Liihe, dies in the former USSR have shown that P. truncatum 1908; may be more prevalent (KHAMIDULLI et al., 1991). At B) Fasciolidae: Fasciola hepatica (Linnaeus, 1758); and any rate, all these diseases are undoubtedly largely unde- Fasciola gigantica Cobbold, 1855; restimated, for different reasons: A) they develop subcli- C) Dicrocoeliidae: Dicrocoelium dendriticum (Rudolphi, nically or only with mild symptoms when parasite num- 1819) Looss, 1899 (= Fasciola lanceolata Rudolphi, ber is scarce, which may be the situation in numerous 1803; = Dicrocoelium lanceatum Stiles et Hassall, cases, so that infected persons do not attend specialists; 1898); Dicrocoelium hospes Looss, 1907; and Eury- B) the diseases they cause can be confused with infec- tretna pancreaticum (1anson, 1889) Looss, 1907. tions of different etiology; C) not all diagnosed cases are All these species have in common the hepatic location finally published. (bile ducts, gall bladder) of the adult stage of the parasite Several of these liver fluke diseases have a geographi- at the level of the definitive (ectopic forms are ho- cal distribution including endemies in several countries wever frequent in humans, mainly in fasciolids), but in in Eastern Europe and in Asia, in which important politi- the case of E. pancreaticum the main location of the cal changes in recent years have led to an increasing adults is in the pancreas and less frequently in the bile opening. That is why these diseases have recently acqui- ducts. red new relevance, efforts being made today by concer- These are the species already confirmed affecting the ned institutions to recognize the serious, yet neglected, human liver, but this does not mean that they are all. problems associated with these trematode infections. There are also other digenean species whose adult stages RIM et al. (1994) appropriately allude to this question are hepatic and whose potentiality to infect humans ap- with the terms of «ignored or emerging» and emphasize pears evident. For instance, Metorchis albidus (Braun, the significant economic impact associated with these 1893) Looss, 1899, an hepatic parasite of carnivorous infections: absenteeism, hospitalization, treatments and mammals in the Holarctic and which is transmitted th- repeated treatments, direct cost to health-care systems, rough rnetacercariae in fish, is considered as a potential disability and agricultural economic losses. source of infection to man, at least in Kazakhstan (SIDO- Moreover, the actual frame can change in the near fu- ROY & BELY AKOYA, 1972) and Lithuania (L1 NIK, ture if appropriate control measures are not undertaken, 1983). Another species, Pseudamphistomum aethiopi- owing to the recently signed GATT international agree- cum Pierantoni, 1942, a member of a genus whose spe- ment, replaced by the World Trade Organization (WTO) cies are all hepatic parasites in mammals (Y AMAGUTI, in 1995. All these twelve fluke diseases are food-borne 1971), has already been found in humans in Ethiopia, infections and the significant increase of international but causing cyst-like nodules in the internal wall of the food exchange which can be expected in the future as the small intestine (CACCIAPUOTI,1947). consequence of facilitated export/import activities bet- Among the human liver fluke species, C. sinensis, O. ween countries can be at the base of infections and the viverrini, O. felineus and F. hepatica are of great medi- appearance of the mentioned diseases far away from the 148 S. MAS-COMA & M.D. BARGUES areas where they are today endemic. This may evidently lations in the recipient countries, it is necessary that ex- concern especially the fish-born trematode diseases, porters in the country of origin assume some responsibi- among which the most important are C. sinensis, O. vi- lity for removing from shipments of plants and or- verrini and O. felineus, all three causing diseases so far namental fish for aquaria and botanical gardens to clearly restricted in geographical distribution (it appears minimize the risk of the spread of snails (MADSEN & to be more difficult in the case of plant- and insect born FRANDSEN, 1989). In the U.S.A, special instructions ). The possibility of geographical range ex- have been issued so that the quarantine authorities may tending in the three opisthorchiid species through fish readily identify exotic land and freshwater snails commerce will mainly depend of the capacity of these (BURCH, 1960, 1982). Such instructions would be useful parasites to adapt to new strains or species, but this on a world-wide scale (MADSEN& FRANDSEN,1989). does not mean that human infections will not be able to occur in non-endemic zones by this way. F. hepatica and D. dendriticum are already good examples of large dis- CLONORCHIS SINENSIS tribution spread to their present wide geographical range, originated through the exportation of living lives- Morphology tock some time ago. The potential banning or importa- tion of fish and fish products from endemic countries The adult stage of the Chinese or Oriental liver fluke, would cause significant economic losses to fish farmers. C. sinensis, is a flat, delicate, transparent, aspinose fluke, All this has already been taken into account by the inter- attenuated anteriorly and somewhat rounded posteriorly, national organisms, which have recently started specific 8-25/1,5-5 mm in size. The oral is slightly larger activities on these questions (RIM et al., 1994). than the acetabulum, which is about a fourth of the way Worth mentioning, finally, is the problem posed in re- from the anterior extremity. A prominent pharynx is fo- cent decades by the spread of freshwater snails, which llowed by two unbranched caeca reaching the posterior act as first intermediate hosts of several human liver body end. The excretory vesicle is long and sigmoid. fluke species, outside and even very far away from the Near the posterior end there are two large branched tes- original endemic zones. Despite the fact that many of tes in tandem, with branches surpassing caeca bilate- these flukes are not very specific at second intermediate rally. The common vas deferens enlarges in the equato- (if any) and definitive host levels (many of these parasi- rial region into a large serpentine seminal vesicle tes use cosmopolitan domesticated mammal species as terminating in a weakly muscular ejaculatory duct which definitive host rather than man), most of them present, opens through the preacetabular, median genital pore. like digeneans in general, endemic areas with well deli- Cirrus pouch and cirrus are lacking. The ovary is small, mited geographical boundaries as the consequence of median, pretesticular and trilobed. The seminal recepta- their marked specificity for their respective first interme- cle is large and transverse, located just behind the ovary, diate snail host species. Among molluscan species invol- outside the caeca. The vitelline follicles are small, dense, ved in the transmission of human liver flukes, this phe- bilateral and confined to the equatorial third of the nomen of spread mainly concerns lymnaeid and thiarid worm. The uterus ascends intraceacally in broad, tightly species (MADSEN& FRANDSEN,1989; POINTlER& MC packed loops, to the genital atrium. The eggs are broadly CULLOUGH,1989), and appears to be related to the ex- ovoid, thick-shelled, light yellowish-brown, with a large, tensive trade in freshwater aquatic plants and aquarium convex operculum, which fits into a rimmed extension fish (MADSEN & FRANDSEN, 1989). The geographical of the eggshell. At the abopercular end there is fre- spread of fascioliasis (there is no second intermediate quently a small tubercular or comma-shaped protube- host in the life cycles of Fasciola spp.) and its relations- rance. Mature eggs are embryonated when laid and mea- hips to the spread of lymnaeid species is already well sure 26-35/12-19 urn (average 29/16 pm). known (BORAY, 1978). In the case of opisthorchiids, transmission foci outside of the original endemic zones Location and definitive hosts have not been found so far, but the spread of thiarid snails must be taken into account, owing to the fact that The adult stage of C. sinensis inhabits the bile ducts, the other hosts involved in their life cycles (fish; cats, gall bladder (mainly in heavy infections) and occasio- dogs and even cosmopolitan peridomestic rats) are avai- nally the pancreatic duct of man and fish-eating mam- lable and can be colonized by the flukes everywhere. mals. Man appears to be the most suitable definitive The world-wide necessity of enforcing drastic measu- host. The natural definitive hosts other than man are dog, res is evident. In certain countries, like Australia and the hog, cat, wild cat, marten, badger, mink, weasel, and rat U.s.A., a very stringent policy has been followed in or- (Rattus norvegicus) and also camels. Among them, stu- der to prevent the import of undesirable and dies in natural transmission foci have demonstrated that plants. An important and effective measure is rigorous dogs, cats, pigs, and rats constitute effecti ve reservoir inspections of shipments of aquarium fish and plants, but hosts. In some regions where prevalence of infection even so lymneids were able to pass the controls in Aus- among humans is very low or lacking, as in some parts tralia (BORAY, 1978). In addition to the quarantine regu- of China, prevalence of infection is usually high among Human liver flukes: a review 149 these mammals. Concerning experimental susceptibility, frozen, dried, or pickled fish shipped from endemic areas guinea pigs are the most susceptible, rabbits, rats, hams- probably accounts for infection in persons who have ne- ters, dogs are relatively susceptible, and mice are less ver left the Hawaiian Islands (BlNFORD, 1934). susceptible. Several bird species have also been found The second aspect is mainly related to human cases re- infected, both naturally and experimentally in the gall ported in several other countries all around the world, bladder (Y AMAGUTI, 1975). even so far as for example the USA(CATA ZARO& Mo- SER, 1982; DRINKA & SHEEHY, 1985; YANGCO et aI., 1987; PAPlLLO, LESLlE& DEAN, 1989; NISHIOKA & Reports in humans Do NELLY, 1990; ONA & DYTOC, 1991), Canada (MC C. sinensis is an important parasite of man in the far SHERRY, 1981; COLQUHOUN & VISVANATHAN, 1987), eastern part of Asia, around the Sea of Japan, East China Hawaii (O'LEARY, BERTHIAUME & SAKBUN, 1985), Pa- Sea and South China Sea. Human cases have been repor- nama (CALERO, 1967), Surinam (OOTSBURG & SMITH, ted from nine countries in Asia, with an estimated 290 1981), Brazil (LEITE et al., 1989), France (BEN-lsMAIL million people at risk and seven million infected (RIM et et aI., 1982; LUONG DINH GIAP et al., 1983), Saudi Ara- al., 1994). Humans of China, Taiwan, Korea, Japan, bia (AL-KARAwl & QATTAN, 1992) or Australia (ATT- Hong Kong, , , Kampuchea (= Cambodia), WOOD & CHOU, 1978), etc., all being imported cases re- and the Far East of Russia, are more or less usually de- lated with travelling persons (immigrants, refugees). tected as infected with the parasite. Although is frequently diagnosed in Orien- Human clonorchiasis is truly endemic at least in tals in the Western Hemisphere, up to the present there is China, Taiwan, Korea, Japan,Vietnam and the Far East no evidence that the infection has become established in of Russia, where the first and second intermediate hosts, regions outside the endemic Asian area. This does not and thus real transmission foci, are found and where the mean an absence of risk of geographical spread of C. si- population is accostumed to eating raw fish.The relative nensis. Asian thiarid snails, including species such as frequency of human cases reported in other countries is Thiara granifera and Melanoides tuberculata, which are related to two epidemiologically important aspects of the known to act as first intermediate snail hosts of this liver parasite biology: the marked resistance of the metacerca- fluke, have undergone an enormous geographical spread riae in fish (they withstand certain types of preparation in the last decades and have successfully colonized seve- of fish, such as salting, pickling, drying, and smoking) ral regions in other continents (MADSEN & FRA DSEN, and the very long life span of the adult stage in man. 1989; POINTIER &Mc CULLOUGH, 1989). This pheno- The first aspect explains most cases found in countries menon, added to the lower specificity of the parasite at neighbouring the true endemic region. In Hong Kong the levels of second intermediate fish host and definitive human infection is known to be highly endemic, but mammal (dogs, cats, pigs, rats) host, gives rise to the neither the snail nor fish intermediate hosts are indige- possibility of geographical spread of the parasite. There nous to the area. Infected fish originating from China are even localities such as Sao Paulo, in Brazil, where and shipped in daily provide the Hong Kong population persons having originally acquired the infection in the with Yu shun Chuk, raw fish congee (BUNNAG & HARI· endemic far eastern Asian region have been detected ASUTA, 1984). Several years ago, reports in Hong Kong (LEITE et al., 1989) and an intermediate snail host spe- told about a prevalence in one fourth of the population cies (M. tuberculatai has already been established after (GIBSON & SUN, 1971) or up to 65,5% (HOU & PANG, several years (V AZ et al., 1986). 1964), and even today clonorchiasis is the most impor- tant (Ko, 1991), being more common in Geographical distribution wealthy people than in low-wage earners owing to the high price of imported fish. In recent years, however, the The geographical distribution of C. sinensis appears to prevalence seems to have decreased because of control coincide with the distribution of the main intermediate measures applied in China (CROSS, 1984; CHA ,1988). snail host species Parafossarulus manchouricus and clo- Persons originating from Laos (BE -ISMAlL et al., sely related hydrobiid snail species. Accordingly, C. si- 1982; CATANZARO & MOSER, 1982; LUONG DlNH GIAP nensis is present in China, Taiwan, Korea, Japan, Viet- et al., 1983; DRINKA & SHEEHY, 1985; O'LEARY, BERT- nam and the Far East of Russia. In this wide geographical HlAUME&SAKBU ,1985; YANGCOetal., 1987;SHERet zone, the incidence of clonorchiasis varies considerably al., 1989; PAPILLO, LESLlE& DEAN, 1989; ONA & DY- from one district to another, even within small regions TOC, 1991) and Kampuchea (= Cambodia) (CA TANZARO (RIM, 1982a). & MOSER, 1982; BEN-IsMAIL et aI., 1982) are more or In China, the present situation of clonorchiasis shows less usually found infected with C. sinensis and detected a distribution in 24 provinces, municipalities and auto- as immigrants in other countries. Clonorchis infections in nomous regions. Studies have shown that the prevalen- persons of Thailand, Malaysia, Singapore and Philippi- ces range between I and 57%. In southern China the re- nes (CROSS & BASACA-SEVILLA, 1984) also seem mainly gions neighbouring Hong Kong and in front of Taiwan, acquired through fish imported from endemic countries, such as Guangdong and Guangxi Zhuangzu, and in nor- but also acquired while visiting there. Consumption of thern China the regions situated north of Korea and bor- 150 S. MAS-COMA & M.D. BARGUES

dering Russia, such as Heilongjiang, Jilin and Liaoning, less than 1000 eggs/g of faeces and without clinical ma- appear to be the zones with highest infection (L1, 1991). nifestations (RIM, 1986). The incidence of infection has In Taiwan, the prevalence and distribution of human been markedly reduced among the under-40 age group clonorchiasis in the periods of 1954-1959,1960-1963 and during the past few years. The decrease is attributed to 1963-1969 were reviewed by CHOW (1960), KIM & the low snail population in these endemic areas due to Ku TZ (1964) and CROSS(1969), respectively. In these industrialization, insecticide pollution of water, land re- years, human infections were reported from nearly every clamation, and health education (Bu NAG & HARI A- county on the island, the prevalence rates varying conside- SUTA, 1984; RIM, 1982a, 1986). rably. According to CROSS(1984), CHEN& HSIEH(1984), In Vietnam, the most heavily infected area is found in RIM(1986) & CHE(1991), today the disease continues to the northern part of the country, in the delta of the Red be found in almost every city/county, with the human in- River, embracing the counties in and around Haiphong fection rate ranging between ° and 57%, human preva- and Hanoi, with prevalences reaching up to 73% in early lence usually being 20-50% in heavy endemic areas. Stu- studies (RIM, 1986). More recently, in Ha Nam Nin Pro- dies in recent years show that the disease is extending to vince, prevalences were of 37,2% in men, 18,3% in wo- new endemic localities in which human prevalences of up men, and as high as 25,8% in the intermediate snail host to 10-20% are found. Rats, cats, dogs and above all pigs Melanoides tuberculata (KIE, BROSHTEI & FAN, develop the role of main reservoirs on the island. 1990). The sporadic cases of clonorchiasis in South In Korea, general estimations speak of 4,5 million in- Vietnam with clinical records were all people from the fected people (BUNNAG&HARI ASUTA,1989; HARI A- orth (RIM, 1986). S TA,PUNGPAK& KEYSTO E, 1993). Endemic areas are In Russia, human cases have been reported in the far scattered all over the country and the most extensive and eastern part, namely in the Amur River Territory and intensive endemic regions are found mainly along the Khabarovsk Territory. Infection rates in snails, fish and Nakdong River and the lower reaches of the other rivers. cats were 2,9%, 9,5% and 74,6%, respectively (POSOK- Several years ago, prevalences and parasite burden rea- HOV, 1982; POSOKHOV,DOVGALEV& BRYU ETKI A, ched up to 82,9% and 10698 eggs/g of faeces in certain 1987; KORABLEV& KOL'TSOV, 1992). areas of the Gimhae Gun county, the heaviest endemic area in the delta of Nakdong River, near Busan, the most Life cycle southeastern part of Korea (RIM, 1982 ). A survey be- tween 1973 and 1982 revealed high infection rates C. sinensis follows a triheteroxenous life cycle which among people living in the basins of the 6 main rivers, develops in fresh water. The adults deposit the eggs in Han, Gum, Nakdong, Mangyong, Yongsan and Seomjin. the biliary passages of the definitive mammal host. Eggs In people living away from the rivers infection was are excreted with faeces after passing through the intesti- usually low (SONG, LEE & RIM, 1983).More recently, nal tract. Egg production (eggs/day/worm) varies with according to RIM(1990), Joo & HOG(1991) and MAu- the kind of definitive hosts: 2400 in cats, 1600 in guinea RICE(1994) it seems that prevalences are slowly decrea- pigs, and 1125-2000 in dogs, 4000 in rabbits. Although sing due to mass treatment programmes. In the Pohang the eggs contain fully developed miracidia, they do not industrial area, along the Hyusnag River, endemicity hatch upon reaching water. The miracidium hatches only markedly decreased compared with previous data, but after ingestion by suitable fresh water snail species. The remains endemic (KIM, HAN, PARK, LIM & HONG, miracidia hatch out in either the intestine or the rectum 1990). In the vecinity of River Ahnseong, prevalence of the snail, then penetrate the digestive wall and be- was 11,0% and, according to age, it showed an increase come sporocysts in the peridigestive regions within 4 followed by a decrease (100 & Ho G, 1991). hours after infection. In Japan, KOMIYA& SUZUKI(l964b) enumerated the The sporocyst is oval-shaped and measures about widely distributed endemic areas: Sendai Plain with the 90/65 urn. It gives rise to cercariogenous rediae, which basin of the Kitagami River as its center; Noshiro area escape from the sporocyst to locate themselves in the around the pond of Asanai-numa; the basin of the Tone connective tissue around the posterior part of the snail River; Lake Kasumigaura and its vecinity; the Kanbara oesophagus, but also in other parts of the snail, 14-17 area; Kaga Plain; the basin of Lake Suwa; Nobi Plain; days after feeding. Rediae are almost sausage-shaped the eastern area of Lake Biwa; the coastal area of Ko- and increasing in size according to development (0,35- jima Bay; the downstream area of the Yoshino River; the 1,73/0,08-0,13 mm). From 5 to 50 cercariae are produ- basin of the Onga River; and the basin of the Chikugo ced inside each redia. The mature cercaria is of pleuro- River. Areas where it was only sporadically found were lophocercous type, with a spinous body wall carrying a Hyogo, Toyama, Fukui, Hiroshima, Ehime, Kawaka, number of slender hairs, two pigmented eyespots, pene- Kochi, and Kumamoto Prefetures. The review by Ko- tration glands consisting of four inner and three outer MIYA (1966) demonstrated that prevalences had progres- pairs, and a tail with a membranous keel on its dorsal sively decreased through time, from a 44,4% infection in and ventral surface (RIM, I982a). The cercarial body is 1917 to 26,0% in 1948. Worth mentioning is that in Ja- of 130-260/43-80 urn and the tail of 258-490/28-53 urn pan, the most infected persons show light infections with in size (YAMAGUTI,1975). Human liver flukes: a review IS I

Cercariae escape from rediae and from the snail hosts, Farn. Thiaridae: Thiara granifera and Melanoides tuber- to become free swimming in water. They are positively culata; Fam.Assimineidae: Assiminea lutea; Fam. Pleu- phototactic and geotropic. The longevity of the cercaria roceridae: Semisulcospira libertina. in water is about 24 hours at 12-27° C and 28-29 hours at The hydrobiid snail P. manchouricus, 7-10 mm in 8-9° C, time period in which the free swiming cercaria height, is the main first intermediate host in all the en- perishes unless it encounters a fish. The cercaria penetra- demic regions where full transmission of the parasite is tes beneath the scales and, losing its tail, encysts as an accomplished, such as in China, Taiwan, Korea, Japan, ovoid metacercaria, chiefly in the muscles and subcuta- Vietnam and the Far East of Russia (RIM, 1982a; PO· neous tissues, less often on the scales, fins, and gills of SOKHOV, DOVGALEV & BRYUNETKINA, 1987; KORA· the freshwater fish (RIM, 1982a). In 5 weeks they deve- BLEV & KOL·TSOV, 1992). All other snail species act as lop into encysted metacercariae, presenting an outer and intermediate hosts in China (RIM, 1982a; LJ et al., an inner hyaline wall secreted by the parasite and a su- 1985). In China, B. fuschsiana is the host in the nort- rrounding capsule formed by the tissues of the fish. The hern part and A. longicornis is also a host in some metacerarial cyst measures 121-160/85-140 urn (KO- areas, but to a lesser extent than P. manchouricus and MIYA & SUZUKI, 1964 a; Y AMAGUTI, 1975). Metacerca- B. fuchsiana. Outside China, S. libertina and T. grani- riae die after 7 hour of desiccation at room temperature, fera may also serve as intermediate hosts in Taiwan 3 min at a temperature of 65° C, and also after 2,5 hourr (CROSS, 1984; CHEN & HSIEH, 1984;CHEN, 1991). B. at 39-40° e. With regard to the duration of the viability chaperi has been reported as the host in the delta of the of the metacercaria, BI FORD(1934) indicated that the Red River in northern Vietnam (GAILLIARD, 1939). metacercariae would remain viable in fish sent from the Also in Vietnam, M. tuberculata has been recently Orient to Hawaii for human consumption. WYKOFF found participating in the life cycle of the parasite in (1958) determined the length of time the metacercariae the Ha Nam Nin Province (KIE, BRONSHTEIN & FAN, remain viable after being taken out of the fish host in Ja- 1990). pan and sent to the USA under refrigeration. An average The two thiarid species, T. granifera and M. tubercu- of 16 days elapsed between the death of the fish and the la/a, have a markedly high colonization capacity, as de- arrival of the metacercariae in Washington, D.e. It was duced from their successful introduction in North (sout- found that from the time of arrival until 40 days after de- hern USA, Mexico), Central (Panama, Caribbean area) ath of the fish host, there was no significant decrease in and South America (Venezuela, Brazil), Europe, Africa, viability, and apparently 50% were viable after 60 days Australia, and Pacific islands, as well as in Asian areas when kept at 3-6° e. other than those of the clonorchiasis enderny (MADSE When the definitive host ingests living metacercariae & FRANDSEN, 1989; POINTIER & MC CULLOUGH, 1989; by consuming raw fish, the parasites excyst in the duo- POI TIER, pers. comm, 1995). The potentiality of geo- denum. The freed migrates to the common bile graphical spread of C. sinensis thanks to this phenome- duct by way of Ampulla of Vater after 4 to 7 hr and then non is evident. to the distal biliary ducts, where it becomes a mature From the ecological point of view, P. manchouricus is worm. In 2 weeks, after their arrival in the biliary ducts, common in ponds, including fish culture ponds, but also the parasites grow to adult stage and initiate their sexual inhabits lakes, swampy areas, and sluggish parts of ri- activity, but it takes another 12 days for the egg to ap- vers and small streams. The other species also have simi- pear in the stools. The prepatent period varies according lar habitats. Studies carried out in Japan have shown that to the definitive hosts (about four weeks in man). About temperature affects the activity of the snails, so that at 3 months are required for the whole life cycle (RIM, low temperatures (10° C), P. manchouricus crawls on 1982a). the mud, but as the temperature rises, it adheres to the The longevity of the parasite is very dependent on the aquatic vegetation for crawling and egg laying (SATO et host-parasite compatibility and the tolerance of the host. al., 1959). In general, the adult stage can survive 15-25 years in hu- In general, infection rates in the snail are low, and mans, but an extreme longevity of over 26-40 years may they show seasonal variation. Of 535 P. manchouricus also be deduced, judging from patients who have long examined from Sun Moon Lake in Taiwan, only 7 resided away from endemic areas (CALERO, 1967; ATT- (1,3%) released C. sinensis cercariae (CLARKE, KHAW WOOD & CHOU, 1978; OOSTBURG & SMITH, 1981; LEITE & CROSS, 1971). In South Korea, in a study from et al., 1989; ISHIOKA & 001 ELLY,1990). March 1979 to September 1980, P. manchouricus was collected in summer (May to August) from localities along the river Taewha, Kyungnam Province, snail po- First intermediate hosts pulation density ranging 10-500/m2 and only 3 Up to nine species of fresh water snails have been re- (0,059%) of 5075 snails (I from each of 3 localities) ported as being able to develop the role of first interme- harboured C. sinensis cercariae (100, 1980). Worth diate host for C. sinensis: Fam. Hydrobiidae: Parafossa- mentioning is the high prevalence 25,8% of the parasite rulus manchouricus (= P. striatulusi, P. anomalospiralis, detected in M. tuberculata in Vietnam (KIE, BRONSH· Bithyniafuchsiana, B. chaperi and Alocinma longicornis; TEIN & FAN, 1990). 152 S. MAs-CoMA & M.D. BARGUES

Second intermediate hosts such as ditches, streams, ponds, and reservoirs in the low flat area. C. sinensis prevalences in large populations of A wide variety of species of freshwater fishes serve as this snail species in many water bodies appears to be the second intermediate hosts, carrying encysted meta- low, but the parasite develops a high intensity of cerea- cercariae of C. sinensis. A total of 113 fish species be- rial liberation from the few infected snails, thus ensuring longing to several families, mostly , has been propagation. Cercariae are shed in the May-October pe- reported so far (RIM et al., 1994). riod in Korea, and in March-October in the more sout- YOSHIMURA(1965) already listed more than 80 fish hern latitudes, as in Taiwan. In Japan, the parasite appe- species so far incriminated as second intermediate hosts ars to be able to survive the hibernation period of the of C. sinensis. Among them, a majority of 71 species are snail under redial stage, cercariae being liberated around confined to the family Cyprinidae, two belong to the fa- April at the time the snail resumes its spring activity. mily Elotridae, and one to each of the families Bagridae, Contrarily, in Korea, cercariae and rediae are fatally da- Cyprinodontidae, Clupeidae, Osmeridae, Cichlidae, Op- maged by the low temperature, so that in such a cold cli- hiocephalidae, and Gobiidae. In addition to these fish mate snails acquire a new infection each year, since the species, certain freshwater shrimp (the crustaceans Cari- resumption of the snail's activity starts in early spring dina nilotica gracilipes,Macrobrachium superbum, and and the cercariae do not start to emerge until late spring Palaemonetes sinensis) have been incriminated as a (RIM, 1982a).The geographical distribution of human source of infection in Nanan, Fukien Province, China endemy is evidently related to that of the intermediate (TA Get al., 1963). The species shown to be infective in fresh water snail hosts. Infected people are found more China, Taiwan, Korea and Japan were critically analysed or less concentrated around fresh water collections by KOMIYA(1966). where transmission takes place.Definitive host preva- In China, about 70 species have been listed, the most lences decrease with the increase of the distance from heavily infected species Ctenopharyngodon idellus, My- these water bodies, although these concer.trations are lopharyngodon aethiops, and Culter alburnus being masked because infected fish can be found quite far used in raw fish dishes as the important food among Chi- away from the snail's habitat and the long viability of nese population (YOSHIMURA,1965; RIM, 1982a). metacercariae in dead fish for human consumption ma- In Taiwan, 13 species of the members of the family kes human infection possible even farther away, not only Cyprinidae and one of the family Ophiocephalidae are from snail habitats but also from fresh water collections. known as intermediate hosts, among which Pseudoras- The long viability of metacercariae also masks the sea- bora parva and Hemiculter kneri (= Cultriculus kneri) sonality of cercarial transmission, which is not clearly are commonly found infected with large numbers of me- transmitted to mammal incidence, although this seasonal tacercariae but they are seldom eaten (KIM & KUNTZ, variation must evidently influence the infection of fish, 1964). In the transmission to humans, Mugil cephalus so that a risk period for the infection of humans and ot- and Ctenopharyngodon idellus are the most important her mammals can be deduced for summer and autumn. ones with above 80% infected, and they are frequently The infection of the definitive mammal hosts takes consumed raw by the inhabitants in endemic areas place by the ingestion of metacercariae together with (CROSS, 1984; CHE & HSIEH, 1984; CHE ,1991). fish food. Every region has one or a few species that are In Japan, 27 species of fishes belonging to the family more infected than all others, and concerning the trans- Cyprinidae (25 species), Gobidae (I species), and Osme- mission to the definitive hosts not all of the fish species ridae (1 species) have been found naturally infected (Ko are of the same importance. The species Pseudorasbora MIYA& SUZUKI,1964), metacercariae being found most parva, owing to its large populations and wide distribu- frequently in the small fishes, such as Pseudorasbora tion as well as to the very high infection prevalences by parva, Sarcocheilichthys variegatus, Acheilognathus metacercariae it is able to support, appears to be the most lanceolata, and Tribolodon hakonensis (RIM, 1982a). important fish in the transmission of the disease, consi- In Korea, about 30 fish species were implicated, most dering all the endemic regions as a whole. frequently Pseudorasbora parva. Sarcocheilichthys si- With regard to clonorchiasis in mammals, fish species nensis, Hemibarbus /abeo, Acanthorhodeus gracilis, involved in the transmission to domestic animals are not Acanthorhodeus asmussi (= Acanthorhodeus taeniana- necessarily the same as those mainly reported for the in- lis), Pungtungia herzi, Pseudogobio esocinus, Gnatho- fection of wild mammals. Concerning domestic cats and pogon atromaculatus, Cultriculus kneri, Macropodus dogs, small fish such as Pseudorasbora parva, Pseudo- chinensis and bidens (RIM, 1982a). gobio rivu/aris and other species, are mainly responsible for transmitting the disease, because they are cheap and consequently used as food for cats and dogs (YOSHI- Epidemiology MURA,1965). The geographical distribution of clonorchiasis is pro- reservoir hosts, including hogs, dogs, cats, nouncedly marked by that of the intermediate snail host rats, and other hosts are found naturally infected, and species, mainly the species Parafossarulus manchouri- their infection intensities do not always coincide with cus. This snail inhabits various types of bodies of water the prevalences in man in the same localities, the inten- Human liver flukes: a review 153

sity of human infection being largely dependent upon Heavily infected small fishes are not generally eaten the eating habits of the population in the place in ques- raw, but they may be eaten after undercooking and may tion. Sometimes similar prevalences appear in animals transmit infection (KOM1YA& SUZUKI, 1964; KOMIYA, and in man, such as in two endemic areas of Korea. In 1966). In China, Cantonese people with a marked prefe- Gimhae Gun prevalences were high in humans (68,8%) rence for raw fish in their diet are notably infected. In and also important in hogs (18,5%), dogs (50%) and central and northern China, however, fish is not eaten house rats (10,9%), whereas in Goyang Gun prevalences raw and there is little or no infection except in certain were lower in humans (15,2%) and also lower in hogs endemic areas, although fish and reservoir hosts may be (2,4%), dogs (21,6%) and house rats (3,8%)(KIM, heavily infected (RIM, 1982a). In the endemic area of 1974). However, sometimes the situation is quite the op- south Fujian Province, China, the residents eat raw sh- posite. In northern China, where little or no raw fish is rimp which is considered to be the only source of infec- eaten except in Cantonese restaurants, prevalences in hu- tion (TANGeta!., 1963; RIM, 1982a, 1986). mans are very low (0,4%) but important in cats and dogs When analysing human infections, three aspects are (25-30%). Tn central China prevalences in humans are worth mentioning: the existence of a social factor, the uncommon but very high in cats and dogs (75-100%). age distribution and the sex distribution. And in southern China prevalences in humans are high The social factor refers to the custom of eating raw fish but not so much in cats and dogs (45%). From all these among the different racial or social groups, this habit ex- and many other data it is evident that reservoir hosts, plaining marked differences of infection among groups, such as cats and dogs as well as pigs and rats, play a sig- such as for instance immigrants with this dietary custom nificant role in spreading the eggs and thus in the trans- in areas where authochtonous people do not have it (KO- mission of the parasite in given areas. However, the in- MIYA, 1966). This social factor is also related to the fa- tensity of human infections is usually heavier than the mily as an epidemiological unit, there being a tendency infection of reservoir animals, indicating that human in- towards familial aggregation (KOMIYA& SUZUKI,1964). fections play a major role in the epidemiology of the di- Concerning age distribution, in various endemic areas sease rather than that of the reservoir hosts (RIM, 1982a, of Japan, Korea and Taiwan as well as in Hong Kong, 1986). the incidence of infection is greater in the higher age Human infection is acquired by ingesting raw, inade- groups and greatest in persons of 30-50 years old, be- quately cooked, dried, salted, or pickled freshwater fish cause clonorchiasis is contracted through eating raw fish flesh which harbours the encysted metacercariae, Con- and the latter represents an adult diet (HoU & PANG, cerning the transmission to man the importance of the 1964; Joo & CHOI, 1974). Contrarily, in some endemic fish species differ, depending from local human diet tra- areas in China where infection is due to the ingestion of ditions. The intensity of human infection is also depen- fish roasted for about 10 min only, clonorchiasis occu- dent upon the eating habits of the population in each rred mainly in children under 15 years of age (Wu, area. Tn most areas, the fish are even raised in fish ponds 1963; SUNG, 1963; KOMIYA,1966; RIM, 1982a). that are commonly fertilized with human and animal fae- Concerning sex distribution, whereas in given areas ces. This provides excellent nutrient for the growth of there is no apparent difference in the incidence of infec- plant and animal life upon which the snails and fish feed, tion in males and females in Japan (KOMIYA, 1966), in and also provides an opportunity for perpetuating the life other areas males have been shown to be more highly in- cycle of the parasite (BUNNAG& HARINASUTA,1984). fected than females, as in Korea (WALTON &CHYU, Fish harbouring metacercariae are frequently small in 1959), China (FAUST & KHAW, 1927; RIM, 1982a), or size and have little or no sale value, depending on the Taiwan (CHOW, 1960). This higher infection rate in men species. For instance, the small species Pseudorasbora is directly related to the habit and frequency of eating parva shows extremely high infections, with 4,44 meta- raw fish, which in turn is probably related to some social cercariae per g and usually hundreds to thousands (a ma- customs.For instance, the Korean people have a traditio- ximum of 31516) metacercariae in a fish (KIM, 1974; nal custom of eating raw fresh water fish after soaking RHEE et al., 1984). them simply in vinegar or red-pepper mash as an appeti- In Japan and Korea, people like to eat raw fish prepa- zer for the drinking of rice wine at social gatherings. red in various ways. Large fish such as Cyprinus carpio Since women infrequently participate in such gatherings, and Carassius carassius are preferred, but sometimes they have much less exposure to infection (RIM, 1982a). also Tribolodon hakonensis. The first two fish species should be considered as the main source of infection, alt- Pathology, symptomatology hough they contain only a few cysts. Similarly in Tai- and clinical manifestations wan, species such as Tilapia mossambica and Ophiocep- ha/us maculatus are commonly consumed raw, but the Human pathology is in the liver, pancreas and spleen. metacercarial infection rates are low. However the repe- Complications are also worth mentioning. ated consumption of raw fish may lead to heavy infec- In the liver, parasites lodge and mature in the medium tions and high incidence caused by accumulated light in- and large intrahepatic ducts, especially those of the left festations due to frequent exposure for several years. liver lobe. Early changes seen in the biliary ducts are ex- 154 S.MAs-CoMA& M.D. BARGUES cessive mucin formation, desquamation, and adenoma- and persist for several months, including malaise, low- tous hyperplasia of the duct epithelium with goblet cell grade fever, high leukocytosis and eosinophilia, an en- metaplasia. In the chronic stage, progressive bile duct larged and tender liver, and hepatic or epigastric pain. thickening dilatation and tortuosity, and ductal and peri- The acute phase is infrequently recognized, and the diag- ductal fibrosis are noted. Biliary stasis results in secon- nosis is difficult to make because eggs may not appear in dary infection leading to pericholangitis, cholangiohepa- the faeces until 3 or 4 weeks after the onset of symptoms titis, pyelophlebitis, and multiple abscesses. Fibrosis (MARKELL& GOLDSMITH,1984). rarely appears in the portal tracts, and portal cirrhosis In chronic clonorchiasis, clinical findings vary accor- has been described only infrequently. Not uncommonly, ding to the worm burden and duration of infection. The the flukes may cause dilation and fibrosis (MARKELL& clinical manifestations appear to increase in severity pro- GOLDSMITH,1984). Principal gross changes are encoun- gressively as the infection becomes older and as the flu- tered in the liver. In massive infections, the organ may kes are gradually acquired. The infection may persist for be enlarged to 2 or 3 times normal size. The external sur- 15-20 years or more. Lightly infected persons usually face of the liver may show pale cystic areas. Linear whi- show no symptoms, but in moderate or progressive cases tish streaks corresponding to dilated bile ducts may be the clinical symptoms show loss of appetite, indigestion, observed. The cut surface reveals a normal parenchyma fullness of abdomen, epigastric distress unrelated to me- punctuated by ectatic bile ducts with walls 2 to 3 times als, discomfort in the right upper quadrant, diarrhea, their normal thickness (DOOLEY& NEAFIE, 1976). Loca- edema, and some hepatomegaly. In heavy infection ca- lized dilation of the bile ducts, usually near the free edge ses the symptoms show weakness and lassitude, epigas- of the liver and especially in the left lobe, is a common tric discomfort, paresthesia, loss of weight, palpitation of manifestation, whereas generalized dilation of the bile heart and tachycardia, diarrhea, vertigo, tetanic cramps ducts is uncommon (GLBSON& SUN, 1971). Parasites do and tremors, and toxemia from liver impairment. In the not invade tissue and thus elict little or no inflammatory later stage of heavy infection or severe cases the symp- reaction. There is a marked increase in mucus produc- toms include marked GI disturbances, with a syndrome tion (DOOLEY& NEAFIE, 1976). associated with portal cirrhosis, splenomegaly, ascites, The pancreas is sometimes also affected. The pancrea- and edema (BELDING, 1965). tic ducts, filled with worms, become thickened and dila- ted and exhibit mucinous and squamous metaplasia. Diagnosis Pancreatitis, usually mild, is however not common (DO- OLEY& NEAFIE, 1976). Splenic congestion is seen in the The parasitological diagnosis is based on coprological early phase of the infection. The spleen may be enlarged, methods for the finding of the characteristic eggs in the showing an increase of fibrous connective tissue espe- faeces or biliary drainage. Care must be taken because of cially in the red pulp in chronic infection with hepatic their small size, and the need to differentiate them from changes (OOSTBURG& SMITH, 1981). eggs of other opisthorchiid and heterophyid flukes As for the complications of clonorchiasis, the occu- which may be present in the same area. Concentration rence of intrahepatic gall stone is one of the most charac- techniques are therefore recommended. The method of teristic pathological features. Cholangitis and cholecysti- formalin-ether sedimentation or other centrifugation tis are caused by bacillary infection during the techniques are more reliable for detecting the eggs in fa- obstructive disturbances of the bile ducts. Liver cirrhosis eces. The direct smear method of recovering eggs from and carcinoma of the bile ducts are often observed in the faeces can be applied in cases of heavy infection, but close association. The evidence for a carcinogenetic ef- in light infections eggs may not be encountered by this fect of clonorchiasis is strongly suggestive. The flukes method (RIM, 1982a). provoke hyperplasia and, in some subjects, neoplasia le- Quantitative methods can be applied to evaluate the cli- ading to cholangiocarcinoma. The association between nical severity. Patients were divided into four groups ac- C. sinensis and cholangiocarcinoma has been documen- cording to the results obtained with the Stoll's dilution ted epidemiologically for many years (RIM, 1986; SHER egg counting method: light infections (1-999 eggs/g of et al., 1989; ONA & DYTOC, 1991). faeces), medium infections (1000-9999 eggs/g of faeces), Only one case of adenocarcinoma of the pancreas as- heavy infections (10000-29999 eggs/g of faeces), and sociated with C. sinensis has been reported recently very heavy infections (30000 eggs/g of faeces and over) (COLQUHOUN& VISVANATHAN,1987). Biopsy of the (RIM, LEE & SEO, 1973). Another quantitative technique, pancreatic lesion revealed well-differentiated ductal ade- the Kato faecal thick smear technique, has also been used nocarcinoma. C. sinensis was detected in the common because of being simple, of low cost and of reproducible bile duct. Following cholecystectomy and choledocho- results, although it is recommended to use it in combina- duodenostomy the patient made an uneventful recovery. tion with other techniques, like the formalin-ether techni- Concerning symptomatology, most patients, even in que (CHAI et al., 1982; LEITEet aI., 1989). endemic areas, are asymptomatic and harbour few para- In patients with biliary obstruction, eggs are not pas- sites. Among symptomatic patients, both acute and chro- sed in stools but may be found in the bile duct by percu- nic syndromes occur. Symptoms and signs may appear taneous needle aspiration. If the patient undergoes sur- Human liver flukes: a review 155 gery, adult flukes and eggs are found in the biliary sys- Treatment tem or pancreatic duct (RIM, 1982a). The immunological tests used for clonorchiasis (indi- The drug of choice is . Doses of 25 mg/kg rect fluorescent antibody test, enzyme-linked immuno- three times daily after meals for 1 or 2 days or 20 mg/kg sorbent assay, indirect haemagglutination test, comple- body weight for 3 days gi ve cure rates of 85 to 100% ment fixation test, counter-immunoelectrophoresis, and (RIM et al., 1982: LOSCHER et al., 1981; WEGNER, others) have been reviewed by HILLY ER (1986). ELISA 1984). The tablets should be taken after a meal, and the has proved to be useful as a screening test, but cross-re- interval between individual doses should not be less than actions with other trematodes (Fasciola, Paragonimus, 4 hours or more than 6 hours. The cure rate of praziquan- ) occur (RIM, 1986). Applied serologic tests tel is related to the intensity of the infection. The cure appear to be generally nonspecific and of little value rate of a single dose of 40 mg/kg was only 22,7-33,3% (Bu ANG & HARI ASUTA,1989). according to the intensity of the infection, although egg In endemic areas where uncooked fish is eaten, clini- reduction rate was high in the patients who were not cu- cal diagnosis is suggestive in patients with an enlarged red (RIM, 1982b). In large scale treatment programmes, liver and symptoms of hepatitis. Advanced infections RIM (1982b) and RIM et al. (1982) proposed a single require differentiation from malignancy, cirrhosis of dose of 40 mg/kg for light infections « I000 eggs/g of the liver, or other causes of hepatic enlargement (RIM, faeces), 2 x 30 mg/kg for moderate infections (1000- 1982a). 10000 eggs/g) and 3 x 25 for heavy infections (> I0000 Haematology can be helpful. Leukocytosis varies with eggs/g). Adverse effects of praziquantel were transient the intensity of infection, and eosinophilia may be pre- and included nausea and vomiting (15%), vertigo (12%), sent. Eosinophilia and liver fuction test in clonochiasis hepatomegaly (4,5%), headache (1,5%), rash (1,5%), were studied by KIM & KIM(1979). and hypotension (1,5%)(YANGCO et al., 1987). Because On transhepatic cholangiograms of the large and me- the drug is also effective in and paragoni- dium-size intrahepatic ducts, the worms appear as cur- miasis infections of the brain, death of these parasites ved filling defects within dilated bile ducts or as mounds may result in larish-Herxheimerlike reactions and se- attached to the duct walls (BUNNANG & HARINASUTA, rious cerebral symptoms, including convulsions, paraly- 1989). Several cholangiography types have been applied sis, coma, or death. Therefore, in areas endemic for both (LEUNG et al., 1989; LIM, 1990), although they are de- clonorchiasis and cysticercosis or , cere- creasing in use because of the introduction of ultrasono- bral involvements must be ruled out before administra- graphy and computed tomography in the diagnosis of tion of praziquantel (BUNNAG & HARINASUTA, 1989). hepatobiliary diseases. Ultrasonography is now conside- The therapeutic effect of is comparable to red of great use for the diagnosis of clonorchiasis, allo- praziguantel. It has the advantage of clearing various in- wing us to determine the parasites in the bile ducts and testinal helminthic infections simultaneously, very low to analyze the extent of the disease at the level of the he- toxicity, excellent tolerance and relatively low cost, alt- patobiliarry tract. It is also useful for the evaluation of hough its treatment course for clonorchiasis needs 7 the effectiveness of a treatment. Ultrasound features of days (LIu et al., 1991). biliary clonorchiasis have been reported by LIM et al. Chloroquine, widely used in the past in treatment, ap- (1989). Because the majority of North American cases pears to inhibit fluke egg-laying only temporarly and is will have light infections and be asymptomatic at the no longer recommended. Hexachloroparaxylene has time of presentation, the abdominal ultrasound is inva- been used in China but is poorly tolerated by many pa- riably normal as are liver function tests (HARINASUTA, tients (BUNNAG & HARINASUTA, 1989). Niclofolan has PUNGPAK & KEYSTONE, 1993). Imaging techniques are also been used and observed to be effective, although the most important in the evaluation of patients with recu- serious side effect affecting the optic nerve indicate that rrent pyogenic cholangitis, an entity that is often seen in it cannot be recommended (RIM, 1990). association with clonorchiasis. The condition is charac- In late or severe clonorchiasis, with gallbladder enlar- terized by dilation and structure of second-order biliary gement and obstruction of the biliary passage, surgery radicals and the presence of intrahepatic pigmented bi- may be useful. In cholangitis with superimposed bacterial liary stones or sludge. Although endoscopic and percuta- infection, antibiotics should be given. Supportive measu- neous cholangiography and ultrasonography readily res such as nutritious diet and fuid and electrolyte control highlight these findings, computed tomography appears may help in recovery (BUNNAG & HARINASUTA, 1989). to be the most sensitive diagnostic modality (FEDERLE, CELLO & LAING, 1982; LIM, 1990). Prevention and control The prognosis is good in those with light infections. Patients rarely die of this infection alone. Death can oc- Measures to control clonorchiasis are directed to re- cur, however, in heavy infections of long standing when duce or eliminate the transmission of the disease, in or- the parasites have caused serious impairment of func- der to prevent new human infections. There are several tion, especially in cases with relapsing pyogenic cholan- ways of control to be applied, according to the different gitis (BUN AG & HARINASUTA, 1989). phases of the life cycle: A) control of snail hosts; B) he- 156 S. MAs-CoMA & M.D. BARGUES alth education; C) treatment of infected persons and do- chiasis was reduced from 27,7 to 19,6% in a period of mestic animals; D) elimination of human and animal fa- about 10 years, health education, as well as cultural, die- eces; E) protection of fish ponds from contamination. tary, and economic changes, appearing to have assisted The choice of the methods must take into account the in the general decrease of infection (CHOI et al., 1977; characteristics of the transmission foci, the habits and RIM, 1979, 1982a). customs of the people, the pattern of transmission, and the resources of the country and endemic area (RIM, 1982a). OPISTHORCHISVIVERRINI Measures to control snail hosts in water collections such as rivers in endemic regions appear to be feasible Morphology with difficulty, but several actions can be applied in man- made water collections such as fish-farming ponds. The The adult stage is flat, elongate, lanceolate, rounded extermination of snails by engineering measures is too posteriorly and attenuated anteriorly, thin, transparent, expensive to be practical, and the molluscicides capable 5,5-9,5/0,77-1,65 mm (mean 7,4/1,47 mm) in size, with of destroying the snails may also destroy fish and other a smooth outer tegument in mature worms. Both suckers aquatic life. However, biological methods could be im- are similar in size. The oral sucker is subterminal and the plemented, such as the introduction of mollusc competi- acetabulum is about one-fourth the body distance from tors or animal predators of the snail host species, trema- the anterior end. The pharynx is small, a short oesopha- tode competitors of C. sine/His such as digenean species gus is present, and the caeca reach almost the posterior able to effectively compete in using the same snail host extremity. The excretory bladder is a long saccular tube species, and sterilizing or pathogenous parasites of the extending up to ovary level. The two testes are markedly snail host species. However, it is still too early to tell. In lobed, in oblique tandem in the posterior body. The long, Japan, among the factors responsible for the marked re- slightly coiled seminal vesicle terminates in a weakly duction of incidence is water pollution from factories, in- muscular ejaculatory duct, which opens through the ge- secticides, and land development with drainage of nital pore immediately in front of the acetabulum. Cirrus swamps, all factors directly affecting snail populations, pouch and cirrus are lacking. The ovary is oval or as well as public health education (KOMIYA& SUZUKI, slightly lobed, median, directly pretesticular. The nume- 1964). Similarly, in Korea, traditional ways are changing, rous vitelline follicles vitellaria are aggregated in a few and the mechanization of farms, the use of chemical ferti- clusters and disposed in two lateral fields in the middle lizer, pesticides, and insecticides may have affected the third of the body. The uterus proceeds anteriad as an in- parasite or its intermediate hosts (RIM, 1979, 1982a). tracaecally coiled tubule which terminates with a metra- The measures to prevent stools containing viable eggs term opening in the genital pore. The eggs are elongate from reaching bodies of water containing the snail inter- ovoid, light yellowish-brown, 22-32111-22 urn (mean mediate host would apply only to the human population, 28/l6 urn; length/width ratio = 1,75) in size, with an since water pollution by reservoir animal hosts cannot be operculum that fits into a thickened rim of the shell pro- controlled. To prevent or reduce infection of the snail per and a minute tubercular terminal thickening (not vi- host, the treatment of the definitive host, mainly humans, sible in all eggs). to destroy the adult worms would be of value as a con- Worth mentioning are the difficulties in differentia- trol measure and appears to be the most feasible in ende- ting O. viverrini from O. felineus, both at adult and egg mic areas. levels. Although differences between adults and bet- At human level, control is evidently related to measu- ween eggs of both species have been found (SADUN, res taken to avoid the ingestion of metacercariae with 1955; RIM, 1982; DITRICH, GIBODA & STERBA, 1990; raw, freshly pickled or insufficiently cooked fresh water HARI ASUTA, Pu GPAK& KEYSTONE,1993), WYKOFF fish.Although complete protection is achieved simply et al. (1965) were unable to distinguish O. viverrlni by cooking fish, it would be a futile exercise to try to get from O. felineus on the basis of morphological characte- millions of people to change centuries-old eating habits. ristics of adult worms or eggs owing to their overlap- Even so, to educate these people to cook their fish would ping intraspecific variability (see chapter on O. felineus not change matters, since fuel is commonly a luxury that for more detai I). At any rate, both Opisthorchis species many cannot afford.At any rate, in the districts where are distributed very far away from each other, so that eating raw fish constitutes a custom, educational activi- the possibility of confusion does not become a problem. ties stressing the importance of thoroughly cooking all This is not the case with C. sinensis, another human li- freshwater fish appears to be the most effective means of ver fluke whose geographical distribution overlaps with preventing clonorchiasis (RIM, 1977). When comparing that of O. viverrini in several countries of southeastern the prevalence rate of clonorchiasis in a certain area in Asia and whose eggs closely resemble those of O. vive- Korea between 1964 and 1976, a marked reduction in rrini in size and shape (RIM, 1982; DITRICH et al., the prevalence was encountered in the youngest age 1992a), so that the possibility of confusion in overlap- group, but there was no significant difference in the ol- ping endemic regions must always be taken into ac- der age groups. The overall prevalence rate for clonor- count. Human liver flukes: a review 157

Location and definitive hosts major survey in seven provinces in the northeast conduc- ted in 1992 indicated that 34% of the human population The adult fluke is a parasite of the distal bile ducts and harbours this parasite. When taking into account local si- also, but less intensively, the gall bladder of man and tuations, prevalences and intensities vary markedly from animals. The civet cat, domestic cat and dog, as well as one community to another. In a very recent survey on 60 other fish-eating mammals, are definitive hosts other villages from 7 Northeast Thai provinces, prevalences than man, which are often found infected with O. vive- detected ranged more or less between a minimum of 8% rrini, even in regions where the infection is not known to and a maximum of 68%, depending on villages and com- occur in humans. In human endemic areas of Thailand, munities. The factors responsible for this variation pro- approximately 60% of the cats and 40% of the dogs were bably include microgeographical characteristics which found to be naturally infected, whereas studies on many determine snail and fish availability as well as recent tre- types of non-domesticated animals never showed parasi- atment programmes and changing eating habits resulting tation by O. viverrini (WYKOFF et al., 1965). from Thailand's extensive public health efforts (SITHlT- Cats, rabbits, guinea pigs and albino rats are used in HAWORN et al., 1994). In central and south Thailand, hu- the laboratory for experimental purposes. These animals man does not appear to be an important are of additional interest because in them the general health problem, prevalences ranging only 0-5% (RIM, pathological changes in the liver are more or less similar 1982; PREUKSARAJ, 1984; HARINASUTA & HARINA- to those in man (RIM, 1982). Rabbits seem to be the best SUTA, 1984). host in the laboratory (WYKOFF & ARIY APRAKAI, 1966). In the neighbouring Laos, the parasite also appears to Dogs can also be experimentally infected, but the worms be common. Because of geographical location and simi- in the liver usually disappear spontaneously after a short lar eating habits, a large number of people are likely to period of time if the metacercariae are not fed repeatedly be infected (WYKOFF et al., 1965). According to the la- (RIM, 1982). Monkeys could not be successfully infected test knowledge, an estimated 1,7 million Loatians are in- (HAR! ASUTA, 1969). Hamsters are easily infected and fected with O. viverrini (RIM et al., 1994). However, are used for immunological studies (SIRISIHA et al., there have been fewer studies in this country. Surveys 1983; CHAWE GKIRTTIKUL & SIRISINHA, 1988), but the carried out in the Vientiane province and its immediate flukes produce relatively few eggs (85 eggs/worm/day) surrounding areas showed prevalences up to 54,4% and (WYKOFF & ARIY APRAKAI, 1966), and the pathological even nearly 100% (BEDIER & CHESNEAU, 1929; SEGAL changes revealed that the liver tissue responses seemed et al., 1968; PATHAMMAVONG, 1971, 1973; SORNMANI to be more destructive and more inflammatory than et al., 1974; GIBODA et al., 1991; PHOLSENA et al., those in other animals (HARI ASUTA, 1969). 1991). In Laos, moreover, high prevalences of minute intestinal flukes (Haplorchis spp.), whose egg morpho- logy is similar, complicate diagnosis (GIBODA et al., Reports in humans 1991). An exact knowledge on the distribution of human O. viverrini infection has been reported in persons infection by O. viverrini in the interior of Laos is today from southeastern Asia, namely Thais, Laotians, Malay- lacking. sians, Vietnamese, Cambodians and Chinese, prevalen- In Malaysia, reports on human infections by O. vive- ces and intensities being mainly related with traditions rrini are limited to the study of BISSERU & CHONG of eating raw or undercooked fresh-water fish. (1969) in the western part of the country. More studies In Thailand, human infection by O. viverrini appears are needed in Malaysia to assess the exact distribution of to be widespread only in the northeastern part and in human opisthorchiasis. some provinces of the northern part of the country (RIM, It is evident that specifically directed surveys are nee- 1982). This progressive and slowly disabling disease is ded to know if human infection by O. viverrini is present considered a public health problem, O. viverrini infec- or not in neighbouring countries such as Vietnam, Karn- tion remaining the leading human parasitic disease in the puchea (= Cambodia) and southern China. in which the northeastern part of the country, with prevalences rea- well-known existence of human infection by C. sinensis ching up to 90% in highly endemic areas (HARI ASUTA, can give rise to confusion due to the difficulties of diffe- 1986). A survey by the Ministry of Public Health in rentiating the eggs of both species. In the neighboring 1984 found an average prevalence of 34% and estimated areas of Vietnam, at least some of the O. felineus human that 7 million of the 19 million people in this region infections reported are thought to represent cases in were infected (PREUKSARAJ, 1984). A more recent sur- which O. viverrini and O. [elineus have been confused vey by the Ministry reported a prevalence of 15,2% na- because of the similarity of their eggs (MARKELL & tionwide, and 24% for the northeast (JONGSUKSANTIGUL GOLDSMITH, 1984). Concerning Kampuchea (= Cambo- et al., 1992), showing a regional drop of 10% over a de- dia) and China, no report on human infection by this spe- cade. However, this survey also detected a pronounced cies could be found in the literature. There are however increase of the prevalence in northern Thailand from several reports on O. viverrini infection in Cambodians 5,6% to 22% in the period 1982-1992, so that conse- and Chinese detected in the territory of Thailand.In this quently the national prevalence did not decrease. A third country, Cambodians and Chinese very rarely eat raw 158 S. MAS-COMA & M.D. BARGUES fish in the traditions of Thais, which explains the low In Laos, up to the present all reports on O. viverrini prevalences by O. viverrini detected in them. SADU concern the same zone, the Vientiane province and its (1955) found in Udorn that only 2,9% of the Chinese immediate surrounding areas (BEDIER & CHES EAU, passed O. viverrini eggs with their stools, whereas 1929; PATHAMMAYONG, 1971, 1973; SOR MANI et al., 50,4% of the Thais did. KEITTIYUTI et al. (1982) found 1974; GIBODA et al., 1991; PHOLSENA et al., 1991), in 0. viverrini eggs in 19% of 5085 Cambodian refugees which the parasite has even been found at larval stage le- hosted in a holding center of the Prachinburi Province, vel in snails, as metacercariae in fish and at adult level in Thailand. cats (GIBODA et al., 1991). Studies are needed to know if Worth mentioning also is the frequency of O. vive- O. viverrini is present in other areas of Laos. rrini infection as reported in studies on human emi- In Malaysia, 0 viverrini has been found in cats (RrM, grants from these endemic countries and carried out in 1982) as well as in humans (BISSERU & CHONG, 1969) other parts of the world, such as Thais in Japan (TA- in west Malaysia. BISSERU & CHONG (1969) also studied NAKA et al., 1988), Laotians and Thais in the U.S.A. the whole life cycle with naturally infected snails in this (HOFSTETTER et al., 1981; WONG et al., 1985; DAO, country, although verification of their result are needed BARNWELL & ADKINS, 1991), Laotians in Germany (DITRICH et al., I992b). Prospections at all levels (snails, (ZIEGLER et al., 1983), Laotians, Vietnamese and Cam- fish, humans, reservoirs) in other parts of this country bodians in France (AMBROISE-THOMAS et al., 1985), are needed to verify if the parasite has a wider distribu- Thais, Laotians and Vietnamese in the Czech Republic tion or not. (DITRICH, GIBODA & STERBA, 1990), or Thais in Ku- In other neighbouring countries in the same area, such wait (HIRA et al., 1987). as Vietnam, Kampuchea (= Cambodia) and the southern bounderies of China, the presence of O. viverrini has ne- ver been reported, except probably the confused deter- Geographical distribution minations of O. felineus in Vietnam (MARKELL & The present knowledge on the geographical distribu- GOLDSMITH, 1984), but cannot be excluded a priori. In tion of O. viverrini shows that it almost overlaps the dis- the latter three countries, the marked similarity of the tribution of the known human infection by this species, egg of O. viverrini with that of C. sinensis, a well-known which can be obviously understood owing to the largely human parasite in Vietnam, Kampuchea and China, does more numerous studies made on the human host. Accor- not make the studies at human level appropriate to disen- dingly, O. viverrini appears to be confined to southeas- tangle the possibility of the presence of O. viverrini. tern Asia, with at least three different countries, in which Specifically directed studies on reservoir hosts such as undercooked fish eating is a more or less usual tradition, cats and dogs (with the possibility to obtain parasite being involved. Reports on this species have been made adults for specific determination after dissection) would mostly in Thailand and only sporadically in Laos and initially be more convenient. Malaysia. Unfortunately, however, in these countries there is an evident scarcity of studies on reservoir hosts Life cycle such as cats and dogs, as well as on the snail and fish in- termediate hosts, which could make possible a more ac- The life cycle of O. viverrini has been reviewed by curate delimitation of the boundaries of the geographical WYKOFF et al: (1965), RIM (1982), HARINASUTA (1969, distribution of this parasite. 1986), HARINASUTA & HARINASUTA(1984), UPATHAM In Thailand, studies have been numerous because of (1988) and HARINASUTA, PUNGPAK & KEYSTONE the recognized medical importance of opisthorchiasis in (1993). The development pattern is typical of opisthor- this country. It has been observed that in the areas of chiids and thus similar to that of C. sinensis and O. feli- high endemicity, man is the most common definitive neus. O. viverrini follows a trihetroxenous life cycle host and thus undertakes the main responsibility for which develops in fresh-water. Eggs already contain ci- maintaining the parasite life cycle in nature. Surveys on liated miracidia when laid by the adults. Thus, fully humans have shown that O. viverrini is more prevalent embryonated eggs are excreted with the faeces of the de- in the northeast and the north of Thailand. In the northe- finitive host, man and other fish-eating mammals. The ast, the reservoir hosts, normally cats and dogs, have an egg-laying capacity of adult flukes in cats is 160-900 infection rate of 40-90%, corresponding with the rates in eggs/adult/day (RIM, 1982) and in humans 2000-4000 man (HARI ASUTA, 1969). But studies on cat and dog (average 3 I60) eggs/adult/day (WYKOFF & ARIYAPRA- reservoir hosts have demonstrated that the geographical KAI, 1966) or an average of 180 eggs/adult/g of faeces distribution of O. viverrini is considerably greater than (ELKINS et al., 1991). Thus, the number of faecal eggs in man. In central Thailand, the life cycle is maintained per gram correlates with worm burden (ELKI S et al., with snails, cats and dogs, although human infection is 199 I; SITHITHAWOR et al., 199 I). Studies in humans not present or found only occasionally at rates of 0-5,0% have suggested density-dependent constraints on fecun- (RIM, 1982). In the south of Thailand, human infection is dity which could operate to restrict the faecal egg output only sporadically found, with a 0,0 I % prevalence (HARI- in heavy infections (RAMSAY et al., 1989;ELKINS et al., NASUTA & HARINASUTA, 1984). 199 I; SITHITHA WORN et al., 1991). Human liver flukes: a review 159

Only those eggs reaching a fresh-water collection, in encysted in cyprinoid fish is probably not longer than one which appropriate aquatic snails are present, will have year (BROCKELMANet aI., 1986). When ingested by hu- the opportunity to continue their development. Once in mans or other fish-eating mammals, the metacercariae water, the inner miracidium must fully mature before excyst in the duodenum or jejunum and migrate through being ingested by a snail belonging to a specific species the ampulla of Vater to the distal bile ducts, where they (CHANAWO G &WAlKAGUL, 1991). After ingestion, attach to the biliary epithelium, mature within 3-4 weeks, the miracidium hatches and penetrates the wall of the and begin to produce eggs (HARlNASUTA,1969). In cats, snail digestive tract to metamorphose to the following rabbits, guinea pigs, and albino rats experimentally infec- larval stage of sporocyst in the peridigestive regions of ted with metacercariae, parasites grow to adult worms in the mollusc. The mature sporocysts are extremely thin- the liver within about 30 days, the size of the worms walled, generally coiled, 1100/650 urn in average size. found in each host species being slightly different depen- This sporocyst stage is already rediagenous. Inner re- ding on the size of the experimental animals (RIM, 1982). diae which have finished their development to mature The entire life cycle requires 4-4,5 months, adult worms stage, escape from the sporocyst. Rediae measure 180- having a life span of 10 years or longer (HARlNASUTA, 1100/80-280 urn (mean 540/120 urn), Mother rediae are PUNGPAK& KEYSTONE,1993). very long, narrow and sac-like, whereas daughter rediae are spindle-shaped or elongate (SCHOLZ,DITRICH& GI- First intermediate hosts BODA, 1992). Rediae migrate to the region of the hepa- topancreas or digestive gland of the snail and begin the O. viverrini appears to be stenoxenous at first interme- production of cercariae. Cercariogenous rediae appear diate host level, with a marked specificity for aquatic in about 1 month. About 15 developing cercariae can be snails of the Fam. Hydrobiidae, Subfam. Bithyniinae be- simultaneously observed within a mature redia (WY- longing only to the genus Bithynia (subgenus Digonios- KOFFet aI., 1965). The prepatent period is approxima- toma) (HARINASUTA,PUNGPAK & KEYSTONE, 1993). tely 8 weeks (CHANAWONG& WAIKAGUL,1991). Four closely related species or subspecies of this genus Cercariae escape from the rediae while still immature have been so far recorded as first intermediate hosts: B. (average length about 200 urn), to continue development siamensis siamensis, B. siamensis goniomphalus, B. sia- up to the cercarial mature stage, which leaves the snail in mensis laevis and B. funiculata.B. funiculata is easily about 2 months. Cercariae shed by the snail are 490- distinguished from B. s. goniomphalus by its more open 565 urn in total length, with a body measuring 140- umbilicus and having a sharp basal keel, whereas B. s. 183/61-96 urn and an unforked tail 350-437 urn long and siamensis and B. s. laevis are mainly distinguished by 26 urn wide on average (WYKOFFet aI., 1965). The body their ecological characteristics. These subspecies of B. of the cercaria is covered with minute spines and at least siamensis should be considered as ecological forms of ten long cilia on each side. It presents a conspicuous pair this snail species (WYKOFFet aI., 1965; DITRICHet aI., of eye spots located anterolaterally to the pharynx, brow- 1992b). Each one of these snails act as first intermediate nish pigment scattered in a bilaterally symmetrical pat- host of O. viverrini in Thailand: B. s. goniomphalus in tern throughout the body, five pairs of penetration glands the northeast, B. funiculata in the north and northwest, with ducts opening dorsal to the mouth, and an excretory and B. s. siamensis and B. s. laevis in the central area of system of 2 [(3 + 3) + (3 + 3 + 3)] flame-cell pattern the country (WYKOFF et al.,1965; HARINASUTA,1969; which remains constant from the time of its emergence RIM, 1982; UPATHAM,1988; DITRICHet aI., 1992 b). from the snail up to its transformation into metacercarial In Laos, B. siamensis goniomphalus has also been found stage. The margins of the tail are drawn out into a thin to be the snail species involved, whereas no infected speci- finlike membrane (WYKOFFet aI., 1965; DITRICHet al., men of B. s. siamensis and B. s. laevis could be found (GI- 1992b; SCHOLZ, DITRICH & GlBODA, 1992). Cercariae BODAet aI., 1991; DITRJCHet al., 1992b), despite CHANA- appear to be both positively phototactic and geotropic, WONG& WAlKAGUL(1991) who demonstrated that B. s. tending to settle and live at the bottom with intermittent goniomphalus is 4-7 times less susceptible to O. viverrini periods of swimming. infection than B. s. siamensis and B.funiculata. When the mature cercariae come in contact with a sui- In Malaysia, BISSERU& CHONG(1969) described the table species of fish, they attach themselves to the scales, natural and experimental infection of a snail species be- lose their tails, and penetrate the tissues, where they longing to the family Thiaridae, Melanoides tuberculata, encyst between the muscle bundles. The metacercarial but, as already discussed by DITRICHet al. (1992b), their cyst is oval in shape, measures 190-250/150-220 urn and parasite identification offers serious doubts (the cercaria is surrounded by a thick layer of host tissue (SCHOLZ,DI- they described do not fit into the characteristics of O. vi- TRICH& GrnODA, 1990). The metacercariae when remo- verrini cercariae). ved from the cyst appear to be oblong in shape, with a size of 310-820/80-210 urn (VAJRASTHlRA,HARINASUTA Second intermediate hosts & KOMIYA, 1961; SCHOLZ,DITRICH & GrnODA, 1990, 1992). In fish, the metacercariae mature in approximately Several species of fish act as second intermediate 6 weeks at 18-20° C. The survival of metacercariae hosts harbouring the metacercariae of O. viverrini. 160 S. MAS-COMA & M.D. BARGUES

In Thailand studies carried out have demonstrated that definitive host species at least in endemic areas, and cats there are not many fish species involved in the parasite and dogs appear to be the main mammal species assu- transmission in nature. The most important species are ring the maintaining of the parasite life cycle in areas Cyclocheilichthys siaja, dispar and with little or no human infection. orphoides, which are prevalent in many provinces. The Although transmission to man may occasionally result other less important species of infected fish are Esomus from drinking water containing metacercariae from de- metallicus, Barbodes gonionotus, Puntius proctozysron, composed fish or from eating dried and salted fish, the P. viehoever, lineatus, and Osteochilus sp. bulk of human infection is acquired through the con- (WYKOFFet al., 1965). sumption of raw or imperfectly cooked fresh-water fish, In Laos, recent studies in Vientiane province have de- which is a common food in all areas of heavy endemi- tected up to seven different cyprinid species harbouring city. Thus, culinary traditions are clearly related to hu- metacercariae: Hampala dispar, H. macrolepidota, Bar- man infection and explain differences of incidences and bodes gonionotus, Puntius brevis (= P. leiacanthusi, prevalences which are detected among people from dif- Puntius sp. 1, Puntius sp. 2, and Cyclocheilichthys re- ferent origins. For instance, Thais, Laotians, Cambo- passon (SCHOLZ,DITRICH& GIBODA, 1990; GIBODAet dians and Chinese inhabiting the northern portion of al., 1991). northeastern Thailand (Udorn) exhibit different eating habits which translate into marked prevalence differen- ces. SADUN(1955) found that 50,4% of the Thais were Epidemiology infected, whereas only 2,9% of the Chinese passed The transmission of the parasite to the fresh-water Opisthorchis eggs with their stools. The Laos descen- snail hosts is related to the defaecating habits of the defi- dent people of northeastern Thailand and of lowland nitive hosts, mainly man but also other fish-eating mam- Laos enjoy traditional dishes prepared from raw or un- mals, especially cats and dogs, in or near fresh-water co- dercooked cyprinoid fish, such as «Koi-pla»,«Pla-sorn» llections where the appropriate snail and cyprinid fish and «Lop-pia». This eating habit constitutes the main species are present. In highly endemic areas of Thailand, mode of human infection by O. viverrini in both Thai- Bithynia snails, especially B. goniomphalus, are always land and Laos. The dish «Koi-pla», the most common available in water bodies. In these endemic areas, the ab- raw fish food eaten and a consuming habit practiced for sence of latrines in villages constitutes an important fac- generations in that area, is eaten immediately after being tor responsible for the propagation of the parasite, owing prepared and usually forms a part of the menu of any ce- to the habit of these people of defaecating on the ground lebration, many of which are held by the local people. in the bush not far from their houses, many of which are This dish is sold in most markets of northeast Thailand situated around the lakes, water beds, or on the banks of and consumed as often as three times a week (SADUN, the streams, so that contamination of the water by the fa- 1955). Another popular food, eaten daily, is raw fermen- eces containing O. viverrini eggs takes place in the rainy ted fish «

the frequency of cholangiocarcinoma is so high within mean breadth of the eggs (1,75 in O. viverrini; 2,75 in O. heavily infected communities that pre-symptomatic ca- felineus) (RIM, 1982, HARINASUTA,Pu GPAK& KEYS- ses could be detected even within small villages. Larger TO E, 1993) although not differentiable by their morp- cross-sectional studies were therefore undertaken by re- hology and absolute measures (WYKOFFet al., 1965). search groups to identify cases of cholangiocarcinoma The sensitivity of the coprological technique is of ut- prior to obstruction, when egg output can be accurately most importance, mainly because of the limitations of all measured (eggs cannot pass through a bile duct comple- techniques to detect very light infections. A comparative tely obstructed by the presence of a tumour). Ultrasound study of three major egg counting techniques indicated and endoscopy were used to diagnose asymptomatic that the formalin ethyl acetate concentration method and cholangiocarcinoma and measure cholangiocarcinoma Stoll's dilution technique were approximately equally prevalence among groups with differing intensity of in- sensitive, while the Kato faecal thick smear technique fection.A number of cases were identified prior to the was markedly less effective in detecting light infections onset of obstruction and jaundice, and therefore levels of (SITHITHAWOR et al., 1994). Furthermore, it has been exposure to the fluke were measurable among those with suggested that as much as 10-15% of infections may be cancer and those without (SITHITHAWORNet al., 1994). underdiagnosed by a single reading using even highly Worth mentioning are the sex- and age-related studies sensitive techniques such as the formalin ethyl acetate which have demonstrated a much higher prevalence of concentration method and Stoll's method (SITHITHA- cholangiocarcinoma among people with heavy infec- WORNet al., 1991). This lack of sensitivity is particu- tions (> 6000 eggs/g) compared to those not excreting larly pronounced when the worm burden is less than 20 eggs. The frequency was moderately elevated among and hence egg output in the faeces is not detectable. those excreting 1000-6000 eggs/g. Interestingly, males Coupled with the nature of fluke distribution in the hu- were more likely to have cholangiocarcinoma than fema- man population, whereby most infections are light, this les, which confirms a sex-associated difference in cancer lack of sensitivity may cause considerable underestima- susceptibility given equivalent exposure. Not surpri- tion in prevalence surveys (SITHITHAWOR et al., 1994). singly, older individuals (>50 years) were more fre- Interestingly, the quantitative faecal egg count by Stoll's quently affected than middle aged (35-49 years) and technique shows a strikingly close positive correlation those aged 24-34 (SITHITHAWOR et al., 1994). with the number of worms recovered in autopsies, indi- But if it appears clear today that O. viverrini infection cating a strong linear association between eggs/g of fae- leads to cholangiocarcinoma, the question arises as to ces and worm burden and thus being useful for the deter- why such closely related flukes as O. felineus or C. si- mination of infection intensity (SITHITHAWORNet al., nensis are not similarly carcinogenic. Possible reasons 1991). Flotation techniques appear to be inappropriate could perhaps be found in aspects such as genetic varia- for the detection of O. viverrini eggs in faeces or conta- tion in the parasites themselves, varying parasite distri- minated soil (HARNNOlet al., 1998). bution patterns, duration of the infection, differences in Although immunodiagnosis would be useful in situa- praziquantel treatment application, other environmental tions in which eggs may not be present in the stool, such factors and host-associated factors such as nutritional as in light infections, biliary obstruction or during the status and genetics. Comparative studies are needed on prepatent period, immunodiagnostic techniques are not these questions (SITHITHAWORNet al., 1994). generally available (HARINASUTA,PUNGPAK& KEYS- TONE, 1993). The indirect immunofluorescent antibody technique gave only relatively satisfactory results (BO- Diagnosis ONPUCKNAVIGK, URATHONG& THAMAVIT,1986).New In the endemic area of southeastern Asia, all patients advances utilizing faecal-based antigen detection are li- complaining of flatulent dyspepsia, pain over the liver kely to facilitate diagnosis in the future. ELISA, mono- region or enlarged liver, and diarrhea or loose stools are clonal antibody-based ELISA, and DNA hybridization clinically suspected to present O. viverrini infection. The are techniques being developed and evaluated for their definitive diagnosis is made by finding the characteristic potential in the detection of O. viverrini infection in hu- eggs in the faeces or duodenal aspirates: Worth mentio- mans (SRIVATANAKULet al., 1985; WONGRATANACHEE- ning is the necessity of a differential diagnosis with C. WI et al., 1988; POOPYRUCHPOG et al., 1990; SIRI- sinensis, whose eggs closely resemble those of O. vive- SIHAet al., 1991;CHAICUMPAet al., 1992). Care must rrini in size and shape (RIM, 1982; DITRICH et al., be taken with possible cross reactions with other human- 1992a) and whose endemic region largely overlaps with infecting flukes present in the O. viverrini endemic zo- that of O. viverrini in southeastern Asia, so that the pos- nes, as already observed by ELKI S et al. (1991) who sibility of confusion in specific determination through found significant associations between antibody levels eggs is evident. Fortunately this is not the case with O. measured by ELISA and echinostome infection. [elineus, from which it can be differentiated by the geo- To determine whether certain tumor markers are ele- graphical separation of the endemic region (human in- vated in Thai patients with cholangiocarcinoma, and fection by O. felineus is known from countries of the old thus might be useful in the diagnosis of cholangiocarci- USSR) and by the ratio of the mean length over the noma associated with O. viverrini infection in Thailand, Human liver flukes: a review 165 the tumor markers CA 125 and CA 19-9 were measured nually in those reinfected), demonstrated that the rein- by radioimmunoassay in serum samples (PUNGPAK et fection rate was 53,9% in the first year and gradually al., 1991). These preliminary results suggest that the me- declined. The findings by MAIRIANG et al. (1993) sug- asurements of CA 125 and CA 19-9 may be useful in the gest that gall bladder abnormalities are reversible follo- early detection of O. viverrini-associated cholangiocar- wing elimination of liver fluke infection with prazi- cinoma. quantel (40 mg/kg), but malignancies, once initiated, Actually, cholangiography, bile examination and ul- are not likely to be affected by treatment. Patients re- trasonography are additional techniques employed for cently treated with praziquantel had higher odds of ab- the diagnosis of O. viverrini infected patients (PUNGPAK normalities compared with others with the same infec- et al., 1989; DAO, BARNWELL & ADKINS, 1991; MAI- tion status who were untreated (ELKINS et al., 1996). RIANG et al., 1992; HARINASUTA, PUNGPAK & KEYS- Satisfactory resolution of morbidity was observed du- TONE, 1993). Portable ultrasonography has proved to be ring two years follow-up after treatment with praziquan- a reliable noninvasive technique in the evaluation of the tel, with significant clinical improvement, normaliza- morbidity due to O. viverrini infection in rural areas tion of laboratory parameters, and downgrading of (Pu GPAK et al., 1997). ultrasonographic abnormalities (PUNGPAK et al., 1997). According to SITHITHA WORN et al. (1994), praziquan- tel utilized in community-based treatment programmes Treatment can help reduce prevalence and intensity of infection, as Trials on Hetol (1,4-bis-trichloromethylbenzole) in well as the frequency of gall bladder disease, but, despite humans showed that the eggs disappeared within 3 we- its widespread use, the frequency of cholangiocarcinoma eks, suggesting that Hetol was highly effective against do not appear to decline and it may be decades before a O. viverrini (HARI ASUTA et al., 1966; Bu AG et al., decline is realized. The recent findings that younger peo- 1970), but this drug was later withdrawn for human use ple, teenagers and those in their twenties, may not be co- by the manufacturers following reports of chronic toxi- operating in control programmes as much as older indi- city on dogs and sheep (RIM, 1982). Later, dehydroeme- viduals, is of great concern, taking into account evidence tine showed to be significantly effective. Oral adminis- suggesting that in some cases malignancy becomes ma- tration of late release tablets of dehydroemetine at a dose nifest 10-20 years after halting infection. If these age of 2,5 mg/kg on alternate days over a 2-month period groups maintain infection into their thirties and forties, gave 84,9% mean egg reduction rate at the follow-up 6 the upcoming generation may not escape the high morta- months after treatment (MUANGMA EE et al., 1974). Ni- lity toll associated with this liver fluke. clofolan, which has a remarkable therapeutic effect on C. Other drugs such as albendazole and mebendazole are sinensis (RIM, 1972; RIM & LEE, 1979), appears to be also effective. Mebendazole at 30 mg/kg for 20-30 days ineffective against O. viverrini infection (RIM, 1982). yielded cure rates of 89-94% (JAROONVESAMA, CHARO- In recent years, praziquantel has become the drug of E LARP & CROSS, 1981), whereas albendazole at a do- choice for the cure of O. viverrini infection. Praziquan- sage of 400 mg twice daily for 3 and 7 days cures only tel is very effective. A 100% cure rate can be obtained 40% and 63%, respectively (Pu GPAK, Bu NAG & HA- with a dosage of 25 mg/kg three times in a single day RI ASUTA,1984) (BUNNAG & HARINASUTA, 1980; AMBROISE-THOMAS, In patients with obstructive jaundice, palliative sur- WEGNER & GOULLIER, 1981). A single dose of 40 to 50 gery in the form of a choledocal jejunostomy is often mg/kg is more convenient for mass therapy and yields a required. In cases with septicemia, cholangitis, or cho- 91-95% cure rate (Bu NAG & HARINASUTA, 1981). A lecystitis, appropriate antibiotics should be given (HA- single dose of 50 mg/kg gave a cure rate of 97% in he- RI ASUTA, PUNGPAK & KEYSTONE, 1993). avy infections (10800-139000 eggs/g of faeces) (PUNG- PAK, Bu AG & HARINASUTA, 1985). A transient but Prevention and control important rise of faecal egg output after praziquantel treatment (from 180 eggs/ml of duodenal juice before The prevention and control measures to be taken in O. treatment to 3486 eggs/ml 12 h after treatment) has viverrini endemic regions are related to the characteris- been observed (RIGANTI et al., 1988). Side effects with tics of the life cycle and should include the control of praziquantel are mild and transient and include diarr- snail hosts, health education, treatment of infected per- hoea (54,5%), dizziness (36,4%), sleepiness (27,9%), sons and domestic animals, elimination of human and epigastric pain (25%), headache (16,2%), nausea animal faeces, and protection of fish-inhabited water bo- (13,2%) and anorexia (Pu GPAK, Bu AG & HARINA- dies from contamination. SUTA, 1985). Although eggs disappear in a week, symp- In recent years, important efforts have been made wit- toms and signs may take a few months to subside hin national control programmes in Thailand, the preva- (DHIENSIRI et al., 1984). An ultrasonographic study ea- lence by O. viverrini having been reduced from 34,6% in rried out by Pu GPAK et al. (1989) in O. viverrini infec- 1981 to 24, I % in 1991 in the northeastern part of the ted patients who had been treated with praziquantel (40 country (RIM et al., 1994). In these control programmes, mg/kg) during 1981-1896 (treatment being repeated an- emphasis has been given and positive results have been 166 S. MAS-COMA & M.D. BARGUES obtained with measures directed toward the changing of be done concerning additional but important control me- the habits of eating raw fish food (especially «Koi-pla», asures which can be implemented at snail, fish and mam- a popular and common food everywhere in northeast mal reservoir host levels. Thailand), teaching the villagers to build and use latri- nes, human treatment campaigns with praziquantel, he- alth education and sanitation improvement. OPISTHORCHIS FELINE US SAOWAKO THAet al. (1993) studied the effecti veness of the intervention measures of giving praziquantel treat- Morphology ment (40 mg/kg) to all infected people either once (vi- llage I) or twice (village II) per year with integration of Opisthorchis felineus is a species very close to O. vive- regular health education and sanitation improvement. A rrini from the morphoanatomical point of view. The control village (village III) received no intervention du- adult stage of O. felineus presents the same general ring the study. The incidence rate per 6 months in the morphological characteristics as O. viverrini. However, two treated villages was lower than that of the control vi- several differences have been distinguished by several llage. Marked improvement in knowledge of opisthor- authors (see review in WYKOFFet al., 1965). O. felineus chiasis, behavioural changes of eating raw fish and in- adults are 8-18/1 ,2-2,5 mm in size and consequently lar- creased numbers of latrines was evident in all of the ger than O. viverrini adults. Another difference can be villages. The study showed that the effectiveness of an- observed at the level of the oesophagus, which is promi- nual drug treatment is similar to that of 6-monthly treat- nent and more elongated in O. viverrini. Concerning ge- ments when combined with regular health education and nital organs, in O. felineus there is a longer distance be- sanitation improvement. FUNGLADDAet al. (1989) alre- tween the anterior testis and ovary, a less marked ady suggested that a primary health care approach can be lobulation of the testes, the location of the posterior tes- used, before and after treatment with praziquantel, as a tis farther from the tip of the caecum, the longer and strategy to control liver fluke infection in rural areas. more winding seminal vesicle, a less lobed ovary, and a An interesting one-year investigation on re-infection distribution of the vitelline follicles not being aggregated rates was carried out by SORNMANlet al. (1984). In vi- in a few clusters. llages where selective population chemotherapy was Eggs can also be distinguished owing to their different combined with improvements in sanitation and health size and shape, those of O. felineus being of 21-36/11- education aimed at changing food habits, the mean 17 urn (mean 30/14 urn). These dimensions clearly over- monthly re-infection rate was 2,0% and the annual cu- lap those of O. viverrini eggs, the difference being found mulative rate was 21,5% whereas in another village, at the length/width ratio, which is 2,75 for O. felineus where only selective population chemotherapy was ca- and only 1,75 in O. viverrini (SADUN, 1955; RIM, 1982; rried out, both rates were 5,0% and 55,5% respectively. DITRICH, GTBODA & STERBA, 1990; HARINASUTA, Another study on re-infection rates after chemotherapy PUNGPAK&KEYSTONE, J 993).Recent studies using made by UPATHAMet al. (1988) showed that subjects electron microscopy have demonstrated that the egg- with high pre-treatment intensities of infection tended to shell has a musk-melon ultrastructure which appears to have heavier intensities of re-infection, about twice that be similar in both species (BEER, GIBODA & DITRICH, of those who were negative or had only light infection 1990; SCHOLZ,DITRICH& GIBODA, 1992). before treatment, indicating that some people are predis- It must be pointed out that WYKOFFet al. (1965) exa- posed to heavy infections. The re-infection rate was mar- mined each character in a large sample and could con- kedly higher than concurrently and previously measured firm that these characters varied considerably, most li- natural incidences of infection. These findings suggest kely representing the extent of contraction at the time of that chemotherapy would have to be applied several ti- fixation. These authors concluded that while some of the mes a year in order to control opisthorchiasis, and that it characteristics (such as the nature of the seminal vesicle) might be more cost-effective to preferentially treat hea- may tend to be present more frequently in one than in the vily infected individuals. other species, the differences appeared to be neither con- Unfortunately, despite the decrease in prevalence and sistent nor specific enough to permit a clear differentia- intensity among age groups over 30 years owing to the tion at adult stage level. Further comparative studies national control programmes, the infection levels still re- with modern techniques (DNA sequencing, isoenzyme main high among the teens and early twenties, which electrophoresis, etc.) are evidently needed in this res- suggests lower levels of participation in treatment pro- pect. Fortunately, there is apparently no danger of confu- grammes and in heeding health education messages. sion thanks to the different, non-overlapping geographi- Special attention should be focused on this group in fu- cal distribution areas of both Opisthorchis species. ture control efforts. Otherwise this age group may re- main at high risk of developing cholangiocarcinoma la- Location and definitive hosts ter in life (SITHlTHAWORNet al., 1994). As can be observed, up to the present all significant ef- The adult stage lives mainly in the larger bile ducts, forts have been made at human level. Much remains to gall bladder and pancreatic ducts. In humans it has an Human liver flukes: a review 167 hepatic location, but in heavy infections worms are also 3) Eastern Siberia: human opisthorchiasis foci have been found in the pancreas (lMAMKULlEY,1971). detected from the Krasnojarsk Territory (GONCHA- It is a natural parasite of fish-eating mammals, mainly ROYA, 1992; ZELYA& GERASIMOY,1992) to the AI- the cat, red, silver and polar foxes, and domestic and tai Territory (NIKITIN& KUIMOYA,1992) and the Ir- wild hogs, but also dogs, wolverine, marten, beaver, kutsk region, at the Lake Baikal (ZHITNITSKAYA et common otter (Lutra lutra), European polecat tMustela al., 1988); putoriusi, Siberian weasel (Mustela sibiricay, sable 4) The basin of the river Kama: situated west of the Cen- (Martes zibellinay, as well as rodents such as the Norway tral Ural Mountains, there is another endemic region; rat (Rattus norvegicus) and Water vole (Arvicola terres- a detailed statistical study of opisthorchiasis carried tris), lagomorphs like the rabbit (Oryctolagus cunicu- out in the Komi-Permyak Autonomous Region, in lus), and pinnipeds such as three species of seals (Phoci- 1985-87 by BRO SHTEI et al. (1989) showed high dae), the Grey seal tHalichoerus grypus), Caspian seal prevalences: 58,3% in man, 79,7-85,5% in fish, 86% (Phoca caspica) and Bearded seal (Erignathus barba- in cats, and 2,5% in snails; Opisthorchis eggs were IUS); in captivity even the lion (Felis leo) has been men- present in 68% of soil and 57% of silt samples; in the tioned (ERHARDT,GERMER& HbR ING, 1962; BOCHA- Kirov region, at the basin of the river Vyatka, a tribu- ROYA, 1976). tary of the Kama river, the parasite was found in 29- 51 % of the cats (burdens 1-519) and 5% of the first intermediate snail host (MALKOY,1991); Reports in humans 5) Central Ural Mountains: in the middle Urals, large Although man may be considered an accidental host foci of human opisthorchiasis (humans: average from the point of view of the life cycle of the parasite, 14,5%, in some foci up to 68%; cats: 83,4%) are this liver fluke is now known to infect approximately found in areas located in the basins of the rivers 1,2 million people in Russia and 1,5 million in the for- Lozva, Sosva, Tavda, Tura and Pyshma (the Tobol ri- mer USSR, according to the latest estimates (RIM et al., ver basin); human and animal infections are particu- 1994). larly high in Serov, Gari, and Tabory districts of the In spite of the large geographical distribution of this Sverdlovsk region (CHURINA, 1973), which in fact species, covering from central Europe (Italy, Switzer- constitutes an endemic zone continuation of the river land, Germany, the etherlands), in the west, to the ex- lrthysh endemic area; treme northern regions, the western Siberian lowlands 6) The basin of the river Volga: it appears that prevalence and Kazakhstan up to the Lake Baikal, in the east (AR- of opisthorchiasis has increased on account of cons- TAMOSHIN& FROLOYA, 1990; ZAYOIKIN, 1991), it is truction of dams and the creation of reservoirs suitable worth mentioning that human infection is not known to for breeding both the snail and the fish hosts, as in the occur east of Poland and the Danube delta (ERHARDT, Rybinsk, Gorky, and Kouybyshev reservoirs (IzyOU- GERMER& HbRNING, 1962; RIM, 1982), although spora- MOYA,1959); human infection is detected also in the dic isolated human cases cannot be disregarded in cen- Volga delta (SEMENOYA& IYANOY,1990); tral Europe (BERNHARD,1985). 7) The Moscow region: autochtonous infection in inha- The disease is endemic with involvement of humans, bitants of the Moscow and Vladimir regions has been domestic animals, and wild animals in an area that co- reported by BRONSHTEI & BEER (1988); vers nearly all the territory of the former USSR, with the 8) Ukraine: there are different foci, the infection rates exception of the northern part of central and eastern Si- varying between 0 and 82%; the Sumy region is a beria, the far-eastern region, the Caucasus, and the for- well-known endemic area of opisthorchiasis, which mer Republics of middle-east Asia (WHO, 1995). Up to comprises the River Dnepr and its eastern tributaries: nine main human opisthorchiasis endemic regions, all in Desna, Sejm, Sula, and Vorskla; another but less im- the former U.S.S.R., can be distinguished: portant endemic zone is the basin of the southern Bug I) Western Siberia: the basins of the rivers Ob and Irtysh river, the Dnestr, and the northern Donets (GRITSAY constitute a very large endemic zone in which the & YAKUBOY,1970); a total of 96 village foci of hu- prevalence of infection reaches 90-95% for man, and man infection were detected in 1984-88 in a vigorous 50-100% in animals (ZAYOIKIN, 1991; ZAYOIKIN, opisthorchiasis programme carried out in the Cherni- DARCHENKOYA&ZELYA, 1991); gov region, at the Desna river, by NESTERENKOet at. 2) Kazakhstan: several zones are endemic, such as the (1990); prevalences of 0-61 % were recorded in the Aktyubinsk, Dzhezkzgan, Karaganda, Pavlodar, Tse- Sumy, Chernigov and Poltava regions (ZAYOIKINet linograd, and Turgay districts; the population at risk is al., 1989b). A review of the geographical distribution 227000 people, and of these the estimated number of of opisthorchiasis in Ukraine has been made by PAD- infected is about 49000 (WHO, 1995); human surveys CHE KO& LOKTEYA(1990); on opisthorchiasis along the Irtysh river in the Pavlo- 9) Bielorussia: foci of opisthorchiasis have been found dar region have shown prevalences up to 14% (GOR- in the Brest, Gornel, and Grodno provinces (WHO, BUNOYAet al.,1988); less important data were recor- 1995); the highest prevalence of infection was recor- ded in the Dzhangeldinsk Region (SMAILOYA,1990); ded in villages in the Dnepr basin and the lowest in 168 S. MAS-COMA & M.D. BARGUES

the Pripyat and Neman river basins (SKRIPOVAet al., neously, positive geotaxis directs them into the deeper 1991). water where these benthophagous fish also usually live Forty-six per cent of the territory of the Russian Fede- (VOGEL, 1934). When the mature cercariae come in con- ration is endemic for opisthorchiasis and nearly 84% of tact with a suitable species of fish, they attach themselves the population of the country reside in these areas. In to the scales, lose their tails, and penetrate the tissues, 1992, opisthorchiasis was endemic in 24 out of 77 admi- where they encyst. These cysts measure 213-2301147- nistrative territories; imported cases were registered in 197 urn and the metacercariae when removed from the 38 others, and in 15 territories no infected people were cyst have a length of 340-590 urn. In fish, the metacerca- found. The only places where there is a risk of acquiring riae mature in approximately 6 weeks at 18-20° C and, infection are the river basins where 10% of the total po- after ingestion by the suitable host, they excyst in the pulation (approximately] 2 million people) reside. The duodenum. When ingested by humans or other fish-ea- health services examine about 200000 people each year; ting mammals, the metacercariae excyst in the small in- 40000-95000 cases of opisthorchiasis were registered testine and migrate to the distal bile ducts, where they annually between 1986 and 1992 in the river basin areas mature and produce eggs (HARINASUTA,PUNGPAK& (WHO, 1995). KEYSTONE,1993).Adults require no less than a month to reach sexual maturity (VOGEL, 1934) and are able to live in the host for 10 years or more. Geographical distribution This species infects several species of fish-eating First intermediate hosts mammals in southern, central, and eastern Europe, i.e. Italy, Albania, Greece, Switzerland, Holland, Germany, Only three fresh-water hydrobiid snail species pertai- Poland, and the European part of the former USSR ning to the genus Codiella have been reported as inter- (Ukraine and Bielorussia), and in Asia it is present in mediate hosts of O. felineus: C. inflata (synonym: Bithy- Turkey, in areas east of the Urals up to eastern Siberia nia inflatai, C. troscheli and C. leachi. C. tentaculata (ERHARDT, GERMER& HORNING, 1962; ZAVOIKl , (considered a synonym of C. leachi by several authors) 1991; WHO, 1995). The distribution of O. felineus in has also demonstrated its capacity to transmit at least ex- freshwater fish and human opisthorchiasis do not coin- perimentally. cide; human infection occurs at some distance from the Among them, C. inflata appears to be the most impor- main endemic areas because of the natural fish migration tant in the transmission. Interestingly, however, cross-in- patterns and transport of fish for sale (WHO, 1995). fection experiments carried out by BEER & GERMAN (1987) demonstrated that there are geographic strain dif- ferences in the compatibility between O. felineus and C. Life cycle inflata. O. felineus follows a fresh-water three-host life cycle, A recent taxonomic review of the Hydrobiidae Bithy- whose development pattern is the same as in C. sinensis niinae considers the group at family level (Bithyniidae) and O. viverrini. Embryonated eggs are excreted with the and redistributes the species in different genera: Bithynia faeces of the definitive host. Once in water, hatching does tentaculata, Codiella leachi, Opisthorchophorus tros- not occur until after ingestion by a snail belonging to a cheli, and Opisthorchophorus hispanicus (syn.: Bithynia specific species. The miracidium hatches and penetrates inflata, Codiella inflata) (BERIOZKINA,LEVINA & STA- the wall of the snail digestive tract to metamorphose to ROBOGATOV1995, ). the following larval stage of sporocyst. Sporocysts deve- lop near the lower intestine and in about 1 month give Second intermediate hosts rise to rediae, which migrate to the region of the digestive gland. The rediae produce cercariae which are shed while The following species of fresh-water cyprinoid fish still immature (VOGEL, 1934). The prepatent period in have been recorded as second intermediate hosts of 0. the snail is from 2 months (VOGEL, 1934) to 4-4,5 felineus: Abramis balerus, A. bramae,A. sapa, Alburnus months (BLYUZNYUK,1963).The liberated cercaria is alburnus, Aspius aspius, Barbus barbus borysthenicus, characterized by pigmented eye spots, ten pairs of pene- Blicca bjoerkna, Carassius carassius, Chondrostoma tration glands with ducts opening dorsal to the mouth and nasus, Cobitis taenia, Cyprinus carpio, Gobio gobio, a flame-cell formula of 2[(5 + 5) + (5 + 5 + 5)] (WYKOFF Leucaspius cephalus, L. delineatus, Leuciscus idus, L. et al., 1965). Its body measures ]32-172/41-48 urn and leuciscus, Phoxinus chekanowskii, P. phoxinus, Polecus the tail is 400-500 um long. The unforked tail has a trans- cultratus, Rutilus rutilus, Scardinius erythrophthalmus parent, integumentary, rudder-like sheath. The cercariae and Tinea tinea (WHO, 1995). are phototactic and geotactic, tending to settle and live at the bottom with intermittent periods of swimming. The Epidemiology positive phototaxis stimulate cercariae emergence from the snail in daylight hours, during the period of maximum The habits of the definitive hosts, mainly man but also activity of fish, their second intermediate host. Simulta- other fish-eating mammals, of defaecating in or near Human liver flukes: a review 169 fresh-water collections where the appropriate snail and the first day of salting, dried in the sun, pickled in garlic fish species are present, allow the infection of the aqua- juice, etc.) is the main factor of opisthorchiasis trans- tic snails by eating the eggs. The snail hosts are infected mission (TIMOCHfNE, 1967; GRITSA Y & Y AKUBOY, by faeces containing the eggs deposited on sandy shores 1970). The greater or lesser importance of given fish and washed into streams. Studies by DROZOOY (1962) species in the transmission varies according to the ende- demonstrated that about 50% of the eggs remain viable mic zones (different prevalences and infection intensi- for a period of 160 days in river water at 0-5° C, and ties in fish, even up to 100% infected - NIKITIN & KUI- thus, many can overwinter in rivers and streams, whe- MOYA, 1992) and human habits. This is reflected in reas in dried faeces they remain viable for only up to 3 human infection. In general, human infection levels de- days at a temperature of -24°C, and for up to 12 days at clined from a downstream to upstream direction, and to- _3° C. According to BEER (1975), the snail infection rate wards the periphery of the basin, where man is least in- in water bodies depends upon the population density of volved in the maintenance of the parasite's life cycle molluscs and the degree of remoteness of the water bo- (ZAYOIKIN, DARCHE KOYA & ZEL YA, 1991). Opisthor- dies from the sources of the invasion. The seasonal dy- chiasis appears to be anthropogenic inasmuch as man is namics of the extensiveness of snail invasion is characte- the main host responsible for the transmission of the di- rized by a one-peak curve. The peak is observed in late sease (ZAYOIKIN et al., 1973). At any rate, in given July, the spring period presenting the greatest epidemio- areas, such as in Kazakhstan, foci of opisthorchiasis dif- logical danger. fer from those in other areas of the former USSR in that Studies by BEER, ZELYA & ZAYOIKIN(1987) on the there are more natural than anthropogenic foci (SIOO- distribution and ecology of the most important interme- ROY & RYBALOYA, 1983). diate snail host species, Bithynia inflata, in the Poltava Carnivores, mainly cats but also dogs, play an impor- region, Ukraine, showed that this snail species is not re- tant role in maintaining the parasite life cycle, showing gularly distributed, but has a mean density of 26 very high prevalences in given opisthorchiasis foci: 76% snails/m", which ranges from 0,1 to 600 snails/m". Its po- of the cats and 70% of the dogs infected in the Altai Ter- pulation density in small rivers was dependent on the ritory (NIKITIN & KUIMOYA, 1992); 61,5% of the local amount of vegetation and on the relationships between cats, with an intensity of 75-560 adults per host, on the floating and submerged plants. B. inflata was absent or river Kama (UCHUATKIN et al., 1988); 29-51 % of the very rare in sites devoid of vegetation or where large cats, with a burden of 1-519 adults per host, in the Kirov above-water macrophytes predominated. Snails infected region (MALKOY, 1991); 68,7-92,8% of the cats in Bie- with O.felineus were found only in waters close to villa- lorussia (SKRIPOYA et al., 1991). ges where infection was present in man or cats. In the Khanty-Mansiisk region, Siberia, human post According to KRIYE KO et al. (1981), O. felineus eggs mortem studies showed that the worm burden was obtained from man, cat, dog, pig, and golden hamster <1000 (in 12 individuals), 1000-5000 worms (in 23), had similar biological properties (size, original viability 5001-15000 (in 22) and >15000 (in 5). Examination of and survival periods in soil and water). The largest faeces showed an irregular distribution of eggs in the amount of infective material was passed by man (82,5 large intestine and a correlation was not established bet- and 96,5% of the total number of eggs shed into the en- ween the number of adults in the liver and the number of vironment of 2 villages), with cats in second place (3,36 egg (BYCHKOY et al., 1990). Also in the Khanty-Man- and 15,8%); dogs and pigs shed relatively small numbers siisk, the prevalence rate was 57,6% in children aged 2 of ova. to 15 and 70,8% for adults. There was a direct correla- The effects of hydrobiological, physical-geographical tion between age-related prevalence levels and the num- and anthropogenic factors on O. felineus infection in the bers of eggs in faeces which rose sharply at the age of 10 population were analysed by PLYUSCHEYA et al. (1990). and reached a maximum level in the 40-year-old Changes in the water regime of a river affect the moll us- (BRONSHTEI , 1985). can hosts and this results in changed prevalence rates in A direct relationship has been noted between infection the human population within the distribution area of the incidence and age. In the Sumy, Chernigov and Poltava intermediate hosts, but not new foci outside it. The crea- regions of Ukraine, human prevalence ranged from 0 to tion of reservoirs in river basins, which are unfavourable 61, I %, so that in children below 14 years old it was 4,2- for opisthorchiasis, results in the desinfection of the re- 21 % and the highest prevalence was recorded in the 30- servoirs in the first 5 to 7 years. Later, during the forma- 50 year old group (ZAYOfKIN et al., 1989b). In Bielorus- tion of the reservoirs, as a result of the restoration of the sia, human prevalence ranged from 0 to 12%, prevalence biotopes in which gastropods develop, an opportunity rising gradually from the age of 14 to a peak in the 25- to for the restoration of opisthorchiasis foci arises (lZYOU- 40-year-old age group (SKRIPOYA et al., 1991). MOYA, 1977). Several studies have demonstrated the interest of hu- Man and other animals acquire the infection by inges- man population migration phenomena on opisthorchia- tion of fish containing metacercariae. O. [elineus infec- sis infection. Concerning immigrants from non-endemic tion is contracted by the consumption of raw or insuffi- areas, the prevalence of infection rises with the length ciently cooked fish (fresh-salted fish, most frequently of of stay in the endemic area, which is particularly mar- 170 S. MAs-CoMA& M.D. BARGUES ked in the first 10 years after arrival and is related to the rely been observed among aboriginal ethnic groups and rapid acceptance of the local custom of eating raw fish indigenous Russians in areas where the prevalence of (BRONSHTEIN,1987). Concerning the consequences of chronic infection is high. Acute opisthorchiasis has, ho- emigration from endemic areas, the risk of dissemina- wever, been reported among new arrivals from other te- ting the disease is related to the long life span of the pa- rritories and occasionally among indigenous Russians rasite adult stage and the presence of appropriate snail who have probably been reinfected after treatment. In and fish hosts as well as with the human diet habits in areas of low prevalence, acute infections are rarely diag- the immigration zones (UCHUATK1Net al., 1988; PUSTO- nosed. No relationship has been found between the pre- VALOVA,1991). sence of acute clinical manifestations and the number of The endemicity of opisthorchiasis in the territories of O. felineus eggs in faeces (BRoNSHTEIN, 1985). Few O. the former USSR is classified according to the preva- felineus eggs are found in the faeces of patients with lence, the intensity of infection, and the degree of clini- acute clinical manifestations. In individuals with low cal manifestations as follows: A) not endemic: imported and moderate liver infection intensity, a positive correla- cases of disease only; B) hypoendemic: sporadic cases tion was established between number of faecal eggs and or a prevalence of < I0%; the mean number of eggs per long-term health deterioration; no such correlation exis- gram of faeces is <100; clinical manifestations are se- ted for individuals with heavy infections (BYCHKOVet vere in <10% of infected persons; C) mesoendemic: pre- al., 1990). Individuals presenting symptoms of early valence is 10-40%; 100-300 eggs are found per gram of opisthorchiasis but not excreting ova have also been de- faeces; clinical manifestations are moderate or severe in tected (ZHURAVLEV& PUZYREV,1987). 10-50% of infected persons; D) hyperendemic: preva- The signs and symptoms of chronic opisthorchiasis in- lence rate is >40%; >300 eggs are found per gram of fae- clude diarrhoea, flatulence, fatty-fodd intolerance, epi- ces; clinical manifestations are moderate or severe in gastric and right upper quadrant pain, jaundice, fever, >50% of infected persons (WHO, 1995). hepatomegaly, lassitude, anorexia, and, in some cases, emaciation and oedema (MARKELL & GOLDSMITH, 1984). Although local damage may be considerable in Pathology, symptomatology the distal biliary tree where the worms are lodged, there and clinical manifestations is usually no measurable effect on liver function. In se- Morbidity and mortality due to O. felineus is very dif- vere cases, hepatitis has been observed (HARINASUTA, ferent from that of its close relative, O. viverrini, alt- PUNGPAK& KEYSTONE,1993). hough signs and symptoms are similar. Authors characte- Most individuals with light to moderate infection rize O. felineus infection as having a number of clinical show no significant signs or symptoms of disease when stages, from acute to late chronic, with accompanying compared with uninfected matched control groups. Pat- symptoms and pathological consequences which can be hological studies have revealed no gross changes in the determined by physical examination (BRONSHTEIN, liver in light or early infections. Epidemiological studies 1986; SITH1THAWOReNt al., 1994). As in other liver fluke have consistently shown that liver enlargement, whether diseases, the degree of its pathogenicity and clinical in- assessed by physical or ultrasound examination, is not volvement depends largely on parasite number and the directly related to intensity of infection. An enlarged duration of the infection. non-functional gall-bladder correlates closely with he- The incubation period between ingestion of metacerca- avy infections. In highly endemic areas, manifestations riae and the appearence of the first symptoms usually va- of chronic infection include cholecystitis, cholangitis, li- ries from 2 to 4 weeks, and rarely is as short as J week. ver abcess, chronic portal hepatitis, hepatic cysts and Maturation of the larval worms in the distal bile ducts ini- gallstones (BRAZHN1KOVA& RODlCHEVA,1989; AL' PE- tiates inflammatory and proliferative changes of the bi- ROVICH,BRAZHNIKOVA& YAROSHKINA,1990;AL'PE- liary epithelium. These changes are accompanied by fi- ROVICH,RODlCHEVA& MITASAOV,1991; RODICHEVA brosis of the distal biliary branches. In heavy infections, & MITASOV, 1991; TUN et al., 1991). In the pancreas a the pathological changes may extend to the proximal bile marked disturbance of external secretion is detected ducts and gall-bladder or be associated with mild peripor- (lMAMKULIEV,1971). Contrarily to what succeeds in the tal fibrosis (WHO, 1995). Adults cause iritation and case of O. viverrini infection, cholangiocarcinoma does trauma to biliary epithelial cells that desquamate and pro- not appear to be associated with O. felineus (PONOMA- liferate, causing glandular or adenomatous formations to REVet al., 1987; BYCHKOV& YAROTSKll,1990). project into the biliary lumen. Periductal infiltration with eosinophils and round cells and fibrosis in the portal areas Diagnosis are commonly found. At this stage, obstruction of biliary tracts occurs with dilatation of intrahepatic ducts and the Diagnosis is based on the recovery of the typical eggs development of subsequent cystic and saccular formations in the faeces or duodenal drainage. Differential diagno- (HARINASUTA,PUNGPAK& KEYSTONE,1993). sis regarding O. viverrini and Clonorchis sin ens is is The acute clinical manifestations are fever, abdominal made using morphological and morphometrical aspects pain, dizziness, and urticaria. Acute infections have ra- (see chapters on these species) but mainly for geographi- Human liver flukes: a review 171 cal reasons, the distribution of O. felineus not overlap- Russians produce a drug called Azinoks, which is an ping that of the other two flukes. analogue of Biltricide (praziquantel) and similar to it in In cases of light infection or biliary obstruction or du- parasitological efficacy and side-effects (STEPANOYA et ring the prepatent period, eggs may not be present in the al., 1991). stool (SADUN, 1955; HARINASUTA, PUNGPAK & KEYS- Other drugs such as albendazole and mebendazole are TONE, 1993). also effective. Albendazole at a dosage of 400 mg twice The Kato-Katz technique (URBAZAEYA & URBAZAEY, daily for 3 and 7 days cures only 40% and 63%, respecti- 1990) and sedimentation techniques with various modi- vely (PUNGPAK, BUNNAG & HARINASUTA, 1984). fications (ZAYOIKIN, PL YUSHCHEYA & NIKIFOROYA, A method for treating 0. felineus infection complica- 1985; PAYLYUKOY, BEREZANTSEY & MEZHAZAKIS, ted by cholangitis is described by AL'PEROYICH, BRAZH- 1990; KOTEL'NIKOY & VARENICHEY, 1991) have been NIKOYA & SOKOLOYICH (1989). This technique involves used for clinical diagnosis of opisthorchiasis in the field the removal of the gall bladder and redirection of the bile and hospitals in the former USSR. Serological tests, into the intestine, with a mandatory external drain of the mainly ELISA, have been used in clinical diagnosis and bile ducts for the purpose of their desinfection, using io- for epidemiological surveys (GlTSU, BALLARD & ZAYOI- dionol or 1% aqueous lugol solution. Daily washing KIN, 1987; GORBUNOYA et al., 1988; ZAYOIKIN et al., ducts should be continued for 10-20 days until no more 1989a; VERBOY et al., 1990). worms or their eggs are present in the bile. Clearing of The reaction double diffusion in agar gel after Ouch- infection in the post-operative period was achieved in terlony is suggested as a diagnostic test in early stages of 50% of 160 patients where traditional disinfectants and human opisthochiasis (RIM, 1982). antibiotics were used, and 90% of 70 patients where ioi- Transhepatic cholangiography may show dilations of dine-containing rinsing agents were used. the biliary tract, classified as mulberry, saccular, or cys- tic, which are considered pathognomonic if found in Prevention and control combination. Occasionally, cholangiography also shows slender filling defects. Leukocytosis varies according to The prevention and control measures are similar to the intensity of infection. Eosinophilia may be present those for C. sinensis and O. viverrini. The infection may (MARKELL & GOLDSMITH, 1984). be prevented by cooking fish and by sanitary excreta dis- posal. The most practical method of preventing human infection is to avoid eating raw, freshly pickled or imper- Treatment fectly cooked freshwater fish. At the same time, the con- Chlorinated derivatives of xylol, such as Chloxyl@ trol of O. felineus infection consists of preventing mainly (hexachloroparaxylol) and hexachlorophene, are very ef- cats, but also dogs and other domestic and free-living po- fective and were extensively used in the past (at a dose tential definitive mammal hosts from eating raw fish. The of 0.15-0.3 g/kg body weight daily, for 2 days) (DROZ- choice of methods must be directed by the nature of the DOY, 1965; PANTYUKHOY, 1966; YALDYGINA TISH- environment, the habits and customs of the people, the CHENKO & MUROMTSEYA, 1971; PLOTNIKOY et al., pattern of transmission, and the resources of the country 1969; SKAREDNOY, 1969, etc.), However, because of its (RIM, 1982). toxicity close medical supervision or even hospitaliza- The strategy of control of opisthorchiasis is based on tion was required, and large-scale chemotherapy was not integration of control activities into primary health care possible (SKAREDNOY & STEPANOYA, 1986). systems, with the main goal of reducing the prevalence The use of these xylol derivates changed with the in- of disease, but in some districts of the former USSR it troduction of praziquantel, which proveed to be very ef- has never been fully operational. In highly endemic fective. A 100% cure rate was obtained with a dosage of areas the achievements of pilot control projects have 25 mg/kg three times in 1 day. A single dose of 40 to 50 been difficult to sustain and the prevalence has usually mg/kg is more convenient for mass therapy and yields a returned to the original levels over a five-year period be- 91 % to 95% cure rate. Side effects with praziquantel are cause people have continued to eat fish that is raw, mild and transient; they include insomnia, headache, slightly salted, frozen or poorly cooked. The migration dizzness, nausea, vomiting and diarrhea. Although eggs of infected fish and consumption by the local people disappear in a week, symptoms and signs may take a even in non-endemic areas contribute to the relative few months to subside (HARINASUTA, PUNGPAK & ineffectiveness of control measures (WHO, 1995). KEYSTONE, 1993). Praziquantel at 75 mg/kg in 3 doses at 4 intervals was well-tolerated and 90,5% effective (BELOBORODOYA, KALYUZHINA & BUZHAK, 1990). FASCIOLA HEPATICA The clinical condition of the patients improved 2 weeks after treatment and 94,3% were free of infection 6 Morphology months after treatment. Symptoms of digestive dysfunc- tion continued in a number of patients following parasi- The adult stage has a broad flat, leaf-shaped body, tological cure (NIKITIN & KUIMOYA, 1992). Nowadays, usually 20-50/6-13 mm in size. The suckers are relati- 172 S. MAs-CoMA & M.D. BARGUES vely small, the ventral being slightly larger than the oral. diagnostic methods, especially in areas where serologi- They are close to each other in a conelike anterior exten- cal tests have been used.The major sources of the infec- sion of the body. The posterior end of the body is bro- tion, domestic herbivorous animals, are widely distribu- adly pointed. The pharynx is prominent and the caeca ted in the world and human infection is not rare in these are long, reaching the posterior end of the body and pre- areas. The disease is mainly endemic in the temperate senting a large number of lateral branched diverticula. and subtropical zones. A moderate temperature and a The testes are also branched, filling the second and third high humidity are necessary for the development and fourth of the body. The cirrus pouch, containing a pro- multiplication of the intermediate snail hosts and the flu- trusible spined cirrus, is well visible, preacetabular, and kes in various development stages. A prolonged, wet opening in a postbifurcal genital pore. The ovary is also summer in Europe has often been followed by an out- dendritic, dextral and pretesticular. The vitellaria are break of the disease (CHEN & MOTT, 1990). dorsal and ventral to the caeca and extend in the whole FACEY & MARSDE (1960) reviewed human infection lateral field of the hindbody. The uterus is relatively by F. hepatica some time ago, and recently. CHEN & short, with several coils situated between the ovary and MOTT (1990) reviewed cases in publications since 1970. the intestinal bifurcation. The eggs are operculated, According to CHEN & MOTT (1990), the following 2594 ovoid, yellow, non-embryonated when laid, and measure cases have been reported during the past two decades: about 130-150/63-90 I1m. They are not readily differen- - Africa:Algeria (6 cases), Egypt (125 cases), Morocco tiated from those of F. gigantica. A mathematical model (I case), Zimbabwe (1 case); for the ontogeny of the F. hepatica adult stage in the de- - America: Argentina (13 cases), Brazil (14 cases), finitive host has recently been developed (V ALERO, Chile (4 cases), Cuba (216 cases), Mexico (5 cases), MARCOS & MAS-COMA, 1996). Peru (163 cases), Puerto Rico (18 cases), Uruguay (16 cases), USA (I case); - Asia: China (41 cases), India (I case), Iran (16 cases), Location and definitive hosts Israel (2 cases), Japan (5 cases), Saudi Arabia (2 ca- The adult stage is a parasite of the large biliary passa- ses), South Korea (3 cases), Thailand (I case), Turkey ges and the gallbladder. This species is a common para- (8 cases), Yemen (3 cases); site of ruminants, especially sheep, goats and , cau- - Europe: Austria (4 cases), Belgium (3 cases), Bulgaria sing important economic losses in the animal husbrandy I case), Czechoslovakia (2 cases), France (963 cases), industry (FROYD, 1975; BORAY, 1981; DARGIE, 1986). the French island of Corsica (2 cases),Greece (I case), A large variety of other domestic and wild animals may Ireland (I case), Italy (1 case), Poland (16 cases), Por- also be infected. The most important alternate hosts tugal (1099 cases), Spain (142 cases), Sweden (2 ca- which play a significant role in the epidemiology of the ses), Switzerland (13 cases), UK (93 cases), former disease are horses, donkeys, mules, and also camelids. USSR (131 cases), West Germany (3 cases), former Wild herbivorous mammals such as as buffalo, , Yugoslavia (I case); wild sheep, wild pig, various marsupials, rabbit, hare, - Australia: 8 cases. and nutria are also susceptible hosts, as well as various According to CHEN & MOTT (1990), of these 2594 ca- wild species in Africa including monkeys. Grazing do- ses, a total of I 103 of the cases have been detected by mestic pigs may also be infected, but this host has a hig- parasitological methods (either by finding the eggs in the her natural resistance against the parasite (BORA Y, stool or bile, or adult worms at surgical operation or at 1982). Many rodent species have been found naturally autopsy), 778 persons have been diagnosed by serologi- infected by F. hepatica and others are usually used for cal tests, 624 persons by parasitological and/or serologi- experimental purposes (MAS-COMA et al., 1987, 1988). cal methods, 28 patients have been diagnosed after pa- thohistological examinations of liver sections, or with ultrasound showing the adult worms, or from their clini- Reports in humans cal persentation, and finally in several cases (61 from Human cases have been reported from numerous Cuba) the diagnostic technique was not mentioned. countries in Europe, the Americas, Asia, Africa and the Worth mentioning is that most papers described small western Pacific (CHE & MOTT, 1990). Several epide- series of hospital inpatients, and only a few community- mics have been recorded in the literature. As the infec- based or epidemiological surveys have detected larger tion may be asymptomatic, and the symptoms and signs numbers of infected persons, as in Peru (STORK et al., are not pathognomonic, the actual number of human ca- 1973), Egypt (FARAG et al., 1979), France (LA BORDE, ses is undoubtedly much greater than the reported. The 1985; RIPERT et al., 1988), Portugal (SAMPAIO SILVA, estimated number of people with fascioliasis is 2,4 mi- CAPRO & CAPRON, 1980; SAMPAIO SILVA, SA TORO llion (RIM et al. 1994). Numbers of clinical cases of F. & CAPRO , 1981) and Puerto Rico (BENDEZU, FRAME & hepatica reported, as well as of infected persons identi- HILL YER, 1982). fied during epidemiological surveys, have been increa- According to the review of CHEN & MOTT (1990), more sing since 1970. These increases may be due to a better than half of the human infections were described in Eu- understanding of the disease and the improvement of rope, mainly in France, Portugal, Spain, the UK and the Human liver flukes: a review 173 former USSR. France is an important endemic area for F. & STEPHE s, 1975; WOOD, STEPHENS & POTER, 1975), hepatica (ANO YMOUS, 1988). The first large modern despite the important livestock production of both and epidemic of human fascioliasis in France occurred in 1956 the high prevalences in sheep and cattle in Australia (COUDERT & TRIOZON, 1958). Between 1950 and 1983, (BORAY, 1969). GAILLET et al. (1983) catalogued 3297 cases from publis- An important paper not included in the review of hed reports.Most cases were reported from the areas of CHEN & MOTT(1990) is that of PICOAGA, LOPERA & Lyon, Bretagne NordlPas de Calais and Sud-Ouest. Wild MONTES (1980), who found 220 human cases in Are- watercress is the main source of human infection in these quipa, Peru, from 1950 to 1977. In Iran, recent estimates areas, where fascioliasis in domestic animals is also suggest more than 30000 human cases (see BAHAR et al., highly endemic. Other recent reports contain detailed re- 1990; MASSOUD, 1990; POURTAGHVA et al., 1990). In views on the situation in South-west France, referring to Spain, a more recent review increases the estimated 274 cases (LA BORDE, 1985) and 37 cases (GIAP, 1987) number of cases between the years 1970 and 1989 to 244 respectively. Most cases have been reported from France, (SORRIBES et al., 1990). On Corsica island, a total of 18 in part because serological tests have been widely used cases were detected between 1984 and 1989 (GIL-BE- there, whereas in other countries the diagnosis of the in- ITOetal.,1991). fections is mainly based on parasitological examinations. Among studies carried out after the review made by The disease is also important in Portugal, with northern CHEN & MOTT (1990), the epidemiological surveys Portugal as a marked endemic area. Cases reported in this made in the Bolivian Altiplano are worth mentioning. country included those in residents from 2 islands, Ma- Although Bolivia is not even mentioned within the re- deira and Cape Verde (ROMBERT & GRACIO, 1984). Con- view by CHEN & MOTT (1990), the Northern Bolivian cerning the former Soviet Union, almost all reported cases Altiplano has proved to be the area in which the highest were from its southern, Asian republic, Tadzhik, near the human prevalences and intensities are known. Different Afghanistan border (KAMARDI OV, 1985; KHASHIMOV & studies have reported human prevalences of up to 70% KAMARDI OV, 1975; RAKHAMA OV, 1987). The situa- in coprological surveys (M AS-COMA et al., 1995; ESTE- tion in Hungary is also worth mentioning, whereas no re- BA et al., I997a, b; ANGLES et al., 1997) and even hig- cent report on fascioliasisis is available from that country her in immunological surveys (HILLYER et al., 1992; despite the several severe outbreaks of human infection MAS-COMA et al., 1995; BJORLA D et al., 1995; recorded between 1959 and 1970, usually after heavy STRAUSS et al., 1997). Intensities in Bolivian children, summer rainfall (KOBULEJ, 1981/1982). There are coun- measured as egg output in stools, ranged from 24 to tries such as Switzerland, in which fascioliasis is quite 5064 eggs per gram (epg), with arithmetic and geometric common in animals, especially in the northern part of that means of 474-1001 and 201-309 epg, respectively (Es- country (ECKERT, SAUERLA DER & WOLFF, 1975), but TEBAN et al., 1997b).Moreover, the human fascioliasis reports on human infection are only occasional. problem in the Altiplano is increased by the presence of In the Americas, Cuba (ESPINO et a/., 1987; FABRE- many other pathogenic protozoan and helminth species GAS RODRIGUEZ et al., 1976; Go ZALEZ et al., 1985; concomitantly parasitizing F. hepatica-infected human GUERRA PEREDA et al., 1980; MILLA MARCELO et al., subjects (ESTEBA et al., 1997a, 1998a, b). 1985; PEREZ RODRIGUEZ et al., 1986; RODRIGUEZ BA- RRERAS et al., 1986) and Peru (KNOBLOCH. 1985; KNO- Geographical distribution BLOCH et al., 1985; STORK et al., 1973) have each repor- ted more than 100 cases. The distribution of the parasite is mainly in temperate In Africa, most cases have been reported from Egypt and subtropical zones, and thus the disease is prevalent (probably due to F. gigantica, since it appears to be the in Europe, North, Central and South America, northern only species in domestic animals in this country) (FARAG Asia, Oceania, and northern Africa and South Africa. et al., 1979, 1986, 1988; FARID et al., 1986; MA SO R et The disease also occurs in some large islands, including al., 1983; RAGAB & FARAG, 1978; SALEM, ABOU BASHA ew Zealand, Tasmania, the UK, Iceland, Cyprus, Cor- & FARAG, 1987). sica, Sardinia, Sicily, Japan, Papua New Guinea, the Phi- In Asia, fewer cases have been described, including lippines, and several islands of the Caribbean. China and Iran (CHEN & MOTT, 1990). In most case re- In Europe, the disease is prevalent in almost every ports from Korea and Japan the parasite was determined county and on adjacent islands, prevalences in animals as Fasciola sp.(AKAHANE et al., 1975;CHO et al., 1976; varying markedly depending on given regions (PANTE- KANEDA et al., 1974; LEE et al., 1982; RIM, 1981; Yos- LOURIS, 1965). In the USA, the parasite is widely distri- HIDA et al., 1974), the question being related to the over- buted but spotty in distribution. F. hepatica occurs most lapping distribution of both F. hepatica and F. gigantica abundantly, with considerable losses among livestock in these 2 countries (CHU & KIM, 1967; RIM, 1981). (MALO E, 1986), in Florida, Louisiana, Texas, Califor- Concerning Australia and ew Zealand, there are only nia, Oregon, Washington, evada, Idaho, Utah, Mon- a few reports from the former (CROESE, CHAPMA & tana, but also in Arizona, New Mexico, Colorado, Arkan- GALLAGHER, 1982; GOODMAN, HE DERSON & CU- sas, Wyoming, Michigan, Wisconsin,Alabama and LLlTY, 1973; MANGOS & MENZIES, 1973; WOOD, POTER Missouri (BORAY, 1982). The potential for the spread in 174 S. MAS-COMA & M.D. BARGUES

Oklahoma has recently been reported (CHERLUYOT& gigantica is dominant, belong to NST. At any rate, AST is JORDA , 1990). The parasite has not established itself in known in Hawaii. NST was also found in Pakistan, the eastern states, other than Florida (BORAY, 1982). Out- Burma, and the former USSR (Vadivostok). Both AST side the continental U.S., the parasite is very common in and NST are however present in several Asian countries: Puerto Rico and Hawaii. It is also endemic in the eastern India, Nepal, Thailand, Vietnam, the Philippines, and Tai- provinces of Canada and British Columbia. In addition to wan. And finally, in Japan and Korea especially the AST Puerto Rico, it is prevalent on several other Caribbean is- is known to be distributed. In the southeastern part of lands, such as Cuba, Hispaniola, Guadeloupe, Martini- Asia, AST flukes are sympatric with ST F. hepatica and que, St. Lucia, and others. In Central and South America NST F. gigantica, but AST flukes are considered repro- it is well known in Mexico, Costa Rica, Venezuela, Peru, ductively isolated from NST F. hepatica and NST F. gi- Bolivia, Brasil, Uruguay, Argentina and Chile (REY, gantica because they perform parthenogenesis. It is clear 1991), as well as in Colombia (MALEK, 1985). that the AST flukes belong to a different strain from those In Africa, it appears in the northern Mediterranean to which NST F. hepatica and NST F. gigantica belong countries such as Morocco and Algeria, where it uses the (TERASAKI,AKAHANE&HABE, 1982). same intermediate snail host as in Europe, Lymnaea This situation has encouraged numerous molecular stu- truncatula (KHALLAAYOUNE et al., 1991; CHEN & dies with interesting results in recent years. Lack of va- MOTT, 1990), as well as in southern Rhodesia and South riation in enzymatic studies has been thought to be due to Africa (PANTELOURIS,1965). However, in Egypt fascio- the parthenogenetic mode of reproduction of these liasis seems to be due to F. gigantica (HAIBA & SELlM, worms, the examined populations consisting of descen- 1960; CHEN & MOTT, 1990). F. hepatica is the species dants of a single individual. True host-induced molecular present at high altitude in Kenya and Ethiopia (BERGEON variation seems rare. A number of authors have commen- & LAURENT,1970). ted on the lack of such variation. In F. hepatica, the same In Oceania, besides New Zealand and Tasmania, it is isozymes were detected regardless of the host species common and widespread in temperate, wet, agricultural (cattle, sheep, goats), although densities of some isozyme areas in the southestern part of the country (BORAY, bands did differ according to host (BLAIR, 1993). On the 1969; SPRATT& PRESlDENTE,1981). island of Corsica, an electrophoretic study on 23 enzyma- In Asia, it is well known from Turkey, Israel, Saudi tic systems allowed the detection of small differences in 6 Arabia, Yemen, the former USSR (to Vladivostok), Iran, systems (AK, CK, EST, HK, MPI, and 6-PGD) between Pakistan, India, Nepal, Burma, China, Taiwan, Thailand, F. hepatica from cattle in the southern part of the island Vietnam, Korea, Japan, and the Philippines (TERASAKI, and from rats in the northern part of the island (PASCUAL AKAHANE& HABE, 1982; CHE & MOTT, 1990). In the et al., 1990). Concerning nucleic acid sequences, studies Asian continent, the distribution of F. hepatica overlaps on ribosomal genes have shown F. hepatica and F. st- that of F. gigantica in various regions, such as in Iran gantica to be distinct, with Japanese Fasciola sp. being and Pakistan, F. hepatica being apparently confined to close to F. gigantica (BLAIR, 1993). high altitude there (KENDALL, 1954; KENDALL& PAR- A recent study carried out by AGATSUMAet al. (1994) FITT, 1959). This overlapping distribution of both spe- has confirmed that Japanese flukes reproduce by parthe- cies has become the basis of an already long controversy nogenesis, regardless of their diploidy, triploidy and rni- on the taxonomic identity of the Fasciola species occu- xoploidy, because of their abnormal gametogenesis. rring in Asian countries, especially Japan, Taiwan, the AGATSUMAet al. (1994) distinguished three different Philippines and Korea, in which a wide range of morp- genotypes among six laboratory-raised, uniparental tri- hological types is detected. At the extremes of this morp- ploid Japanese isolates with no normal sperm formation, hological range, some resemble F. hepatica, whereas ot- indicating that these parthenogenetic lines have arisen hers resemble F. gigantica, with an intermediate form independently of each other: genotype I = F. hepatica- also occurring. like worms; genotype 2 = F. gigantica-like worms; and Studies have shown that the three morphological types genotype 3 = intermediate form worms.Genetically, F. (hepatica, gigantica, intermediate) exist in Japan, two ch- hepafica-like worms were clearly distinct from the other romosome types having been found: diploid (2n = 20) and two genotypes. Other F. gigantica-like worms from Ko- triploid (2n = 30). Another type which had both 20 and 30 chi belonged to genotypes 2 and 3. Korean worms re- chromosomes within a single fluke was also found. In sembled genotypes 2 and 3 more than genotype 1, and both diploid and triploid types spermatogenesis is abnor- differed from American and Australian diploid strains mal and no fertilization occurs. Flukes with a few or no with sperm. American and Australian strains had similar spermatozoa in the seminal vesicle perform abnormal patterns and proved to be mendelian populations. The in- spermatogenesis (called «abnormal spermatogenetic dependent origins of parthenogenetic strains in Japan type» -AST), while those with many perform normal might have occurred through independent hybridization spermatogenesis (called «normal spermatogenetic type» - events between strains. The existence of such hybrids NST). All specimens examined from Europe, South and would explain the continuing confusion among scientists North Americas, and Oceania, where mainly F. hepatica with respect to the taxonomic status of the Japanese liver is considered to be distributed, and from Africa, where F. flukes. Human liver flukes: a review 175

Life cycle contacting a solid support, mostly leaves of water plants above or below the water line. They then lose their tails The general pattern of the diheteroxenous life cycle of and quickly encyst, changing into metacercariae. Meta- F. hepatica was the first to be elucidated among tremato- cercarial cysts are round and about 200 urn in diameter, des and has already been the subject of several extensive and become infective within 24 hours after encystment. reviews (TAYLOR, 1964; DAWES & HUGHES, 1964, Floating infective metacercarial cysts are also originated 1970; PANTELOUR1S,1965; KENDALL, 1965, 1970; no. at the level of the water surface line (VARE1LLE-MoREL, RAY, 1969; OOEN1NG,1971). DREYFUSS& RONDELAUD,1993).Metacercarial cysts Eggs are produced by parasite adults and are excreted are resistant and remain viable for a long period, but are with faeces. Daily egg output per adult fluke is generally killed by excesive heat and dryness. inversely proportional to the intensity of the fluke bur- Metacercariae infect the definitive host after inges- den. In moderate infections the daily egg output is tion. A proportion of metacercariae die in the gastroin- usually constant, but in heavy infections egg output va- testinal tract and a relatively few eventually develop ries considerably (CHEN & MOTT, 1990). The parasite into adults. Metacercariae excyst in the small intestine follows an aquatic life cycle. Eggs mature in water. If within an hour after ingestion, penetrate the host's intes- the climatic conditions are suitable (15-25° C), the mira- tine wall, and appear in the abdominal cavity by about 2 cidia develop and hatch in about 9 days (LAPAGE, 1968) hours after ingestion.Most reach the liver within 6 days to 21 days (BORAY, 1969). If conditions are unfavoura- after excystment. In the liver they migrate for 5 to 6 we- ble, they may not mature but may remain viable for se- eks, preferentially feeding directly on liver tissue. They veral months (LAPAGE, 1968). The miracidium, about eventually penetrate into the bile ducts where they be- 130128 urn, hatchs under light stimulation and swims ra- come sexually mature. The prepatent period is about 2 pidly by means of its cilia until it contacts an appropriate months (6-13 weeks) in sheep and cattle (CHEN & aquatic or amphibious snail host. The miracidium is po- MOTT, 1990). Thus, the whole cycle takes about 14-23 sitively phototropic and negatively geotropic. Miracidia weeks (LAPAGE, 1968; BORAY, 1969). It has also been failing to penetrate an appropiate snail die within 24 speculated that the immature flukes may enter the blood hours (OLSEN, 1974). stream and be carried to various parts of the body, or The miracidium penetrates the snail and changes into may reach the liver by travelling up the bile duct (CHEN an elliptical saccular sporocyst, 150-500 urn in length, & MOTT, 1990). in the mantle, mantle collar and perioesophageal area. The prepatent period (from the ingestion of metacerca- This sporocyst produces mother rediae which in turn riae to the first appearance of eggs in the faeces) varies produce cercariogenous daughter rediae. Mature rediae, according to the host, and also depends on the number of presenting a rudimentary digestive system (pharynx and the adult flukes in the liver, so that the greater the fluke a short caecum), are cylindrical, about 250-750 urn in number, the longer the time to mature and to initiate egg length, with a raised collar near the anterior end and two laying: 35-42 days in mice; SS days in guinea pigs; 63 bulging projections in the posterior third of the body. days in sheep infected with 200 metacercariae, 13-15 They come out of the sporocyst and migrate mainly to weeks in sheep infected with 2000 metacercariae; 56-61 the digestive gland. Up to four redial generations have days in cattle, depending on host age (BORAY, 1969; DE been found, although 3 generations are usually produ- LEON,QUINONES& HILLYER, 1981). In man, a period of ced after a monomiracidial infection (RONDELAUO& at least 3-4 months is necessary for the flukes to attain BARTHE, 1986). The redial generations follow the same sexual maturity (FACEY& MARSDEN, 1960; WASOWA, developmental pattern in different Iymnaeid species AUDRZEJAK& JANICKI,1979). (RONOELAUD& BARTHE, 1987). Cercariae develop wi- Several studies (DAWES& HUGHES, 1964; LAPAGE, thin 6-7 weeks at 20-25° C. At lower temperatures the 1968; SMITHERS,1982) show that the life-span of the pa- development is delayed. rasite in sheep can be as long as II years and 9-12 The cercaria has a large, almost round, spinose body months in cattle. Concerning man, DAN et al. (1981) (28-3201250 um) and a long, simple, motile tail (about suggested that F. hepatica may survive for at least 9 ye- 700 um long). Cercariae escape from the redia through a ars based on imported cases from Afghanistan, and up to birth pore located just posterior to the collar at the ante- 13,5 years according to another report. CHATTERJEE rior end, and are shed by the snail into water. The prepa- (1975) estimated that the life span of the adult fluke in tent period is dependent on temperature, higher tempera- man is between 9 and 13 years. tures reducing the period (15° C: 56-86 days; 20° C: 48-51 days; 25° C: 38 days). The shedding process takes First intermediate hosts place between 9° and 26° C, independently of light or darkness, and it seems to follow an infradaily shedding The intermediate snail hosts are amphibious and pattern of 7 days in the daily production during the aquatic species of the family Lymnaeidae. Principal or whole emergence and a circadial rhythm with maximum obligatory intermediate snail hosts mentioned for F. he- production between midnight and I am. (AUDOUSSETet patica are: Lymnaea truncatula in Europe including al., 1989). Cercariae swim for a short time (1 hour) until most of the former USSR; L. truncatula and L. colume- 176 S. MAs-CoMA & M.D.BARGUES lIa in Africa; L. humilis, L. bulimoides and L. cubensis include frequent drastic environment changes and insec- in North America; L. viatrix (= L. viator) and L. diap- ticide treatments, such as rice fields (V ALERO et al., hana in South America; L. truncatula and L. viridis in 1998b). Asia; L. tomentosa in Australia; L. tomentosa, L. colu- It has recently been shown that computer-based Geo- mella and L. truncatula in New Zealand; L. viridis and graphic Information System (GIS) may be used to cha- L. ollula in Hawaii, Papua New Guinea, Philippines and racterize the epidemiology of the disease, soil-hydrology Japan. Alternate or facultative host species are: L. pa- based GIS models being proposed for analysis and pre- lustris and L. glabra in Europe and former USSR; L. co- dictions (MALONE, 1994). However, such methods do lumella in North and South America; and L. columella not appear today to be sensitive enough to detect the lo- and L. viridis in Australia (BORAY, 1982). Later studies cal patchy distribution of snail populations according to have also shown the role of L. cubensis in Central Ame- environmental elements such as small salt concentration rica and northern South America, and L. gedrosiana in differences in water collections. Also recently, a fascio- Iran (CRuz-REYES & MALEK, 1987). Research studies lid-specific molecular assay has been developed to per- on snail anatomy and shell morphology (OVIEDOet al., mit the study of seasonal transmission patterns and para- 1995; SAMADIet al., 1997), DNA sequencing (BAR- site-snail interactions. This assay detects individual GUES& MAS-COMA, 1997; BARGUESet al., 1997) and infected snails immediately after miracidial exposure isoenzymatic studies (JABBOUR-ZAHABet al., 1997) and throughout the parasite's development period proved that L. truncatula is the only intermediate host (ROGNLlE, DIMKE &KNAPP, 1994), but possible cross- species in the Northern Bolivian Altiplano. Several of reactions with other digenean parasites using the same these snail species are included in other Iymneid genera snail species have not yet been evaluated. by MALEK(1985). The development of the parasite larval stages in the Interestingly, recent molecular biology studies have snails is inversely proportional to the ambient tempera- confirmed the applied parasitological importance of the ture (BORAY, 1969). Although studies have demonstra- 18S rRNA gene, both in the distinction between fascio- ted that parasite development is arrested below 10° C or liasis transmitter and non-transmitter lyrnnaeid snail spe- over 30° C (BORAY, 1969; OLSEN, 1974), recent experi- cies and in the distinction between Iymnaeid species mental research by MAS-COMAet al. (unpublished data) which transmit F. hepatica and those which transmit F. carried out with an F. hepatica strain from the Northern gigantica, as well as in the development of specific pro- Bolivian Altiplano (4000 m altitude) has shown that this bes for the distinction of infected from non-infected parasite is sometimes able to adapt to more extreme con- snail individuals in epidemiological surveys and control ditions and continue its development even when the lo- studies of human and animal fascioliasis (BARGUES& cal daily ambient temperature falls below 0° C and the MAS-COMA,1997; BARGUESet aI., 1997). water temperature decreases to 5° C during the night. Lymneid snails appear to be more resistant to low than to high temperature. They can survive through the winter Epidemiology although there is little or no development and multiplica- Human infection is determined by the presence of the tion (BORAY, 1969). Contrarily, persistent high tempera- intermediate snail hosts and herbivorous animals, and re- tures and dry conditions adversely influence both snail lated to climatic conditions and dietary habits of man. populations and parasite larval stages. The metacercariae Both appropriate snail host species populations and may survive for long periods at low temperatures if the parasite larval stages are dependent on the presence of level of moisture is sufficient, but they are susceptible to the necessary water and of the local climatic conditions. desiccation and to temperatures over 25° C(BORAY, Concerning the ecological characteristics of the snail 1969). In contrast, high humidity associated with heavy species, two recent studies have shown that under spe- rainfall and moderate temperatures may herald hyperen- cial circumstances given Iymneids are really able to demicity in herbivorous animals. Thus, human infection adapt to extreme conditions, thus contributing to the has been more frequently observed in the years with he- spread of the disease. In Corsica, L. truncatula has pre- avy rainfall in France (RIPERT et al., 1988). ferentially adapted to being in reservoir habitats (per- Mainly sheep, goats and cattle act as animal reservoir manent presence and renewal of water) instead of in in- hosts in relation to man. There is no evidence that sheep vasion habitats (only seasonal presence of water) as is or goats acquire immunity against F. hepatica, whereas usual in the European continent, and several atypical cattle are resistant to challenge after initial infections. habitats even suggest an ecological niche-widening, as a According to BORAY (1969), in sheep the egg output of consequence of the influences of the insularity pheno- the adult flukes is relatively high (daily output of 4000- menon (OVIEDOet al., 1992). In Bolivia, the Iymneid 50000 eggs per fluke and 8800-25100 eggs per host in intermediate host is perfectly adapted to the extreme cli- weeks 13-19 after infection), whereas in cattle the dura- matic conditions of 4000 m altitude of the human fas- tion of egg production is short and high egg output lasts cioliasis high endemic zone in the Northern Altiplano for only a few weeks. Most of the flukes in cattle are eli- (MAS-COMA et al., unpublished data). In southern Eu- minated within 9-12 months (BORAY, 1969; DAWES & rope, it is even adapted to places where human activities HUGHES, 1964; SMITHERS, 1982). Thus, sheep play a Human liver flukes: a review 177

more important role in contamination of the pastures and ral disease and sheep- or cattle-herders are more fre- in human transmission. quently infected than those in other professions (STORK A large variety of other domestic and wild animals as et aI., 1973). well as laboratory animals can be infected with F. hepa- Further analysis of the literature allows us to distin- tica, but they are usually not very important for transmis- guish three types of reports (CHE & MOlT, 1990): A) sion of the human disease (CHEN& MOlT, 1990). Howe- the majority of papers concern only individual case re- ver, MAS-COMAet al. (1987, 1988, 1990) and VALEROet ports; B) the incidence of infection is significantly ag- al. (1992, 1998a) have demonstrated, both experimen- gregated within family groups because the family shares tally and in the nature, that the rat Rattus rattus may play the same contaminated food (FARAGet al., ·1979; GA- an important role in the epidemiology of the disease, in LLARDO,SAFZ & E RIQUEZ,1976); C) the existence of the spread as well as in the transmission of the parasite, at only a few reports on community-based surveys having least in the Mediterranean island of Corsica. Moreover, shown a large numbers of infected persons identified by MAS-COMA et al. (1997) have recently concluded that stool examinations combined with serological tests (FA- pigs and donkeys also represent important reservoir hosts RAGet al., 1979 in Egypt; STORK et al., 1973 in Peru; participating in the transmission of the parasite in human SAMPAIOSILVAin CHEN& MOlT, 1990 in Portugal) and endemic areas, such as the Northern Bolivian Altiplano, indicating that symptoms were not pathognomonic nor and have consequently emphasized the need to take pigs were they severe enough for most persons to seek medi- and donkeys into account within preventive and control cal attention. Familial clustering and high prevalences measures against human fascioliasis. Among wild ani- have been also found in community-based surveys in mals, lagornorphs have also shown to be able to develop Corsica and Bolivia, respectively (M AS-COMAet al., un- a role in the epidemiology of the disease in different areas published data). (BAILENGERet al., 1965). Worth mentioning at this point are the results of expe- Pathology, symptomatology rimental research carried out by BARGUESet al. (1996b) and clinical manifestations which demonstrate the viability of humans as definitive host, eggs excreted by infected persons of the Bolivian Little information is available on the pathology offatal Altiplano being able to experimentally start the life cycle fascioliasis (ACOSTA-FERREIRA, VERCELLI-RElTA & of the parasite for several generations in the laboratory. FALCO I, 1979; DUAN et al., 1986) since death rarely According to the investigations made by MAS-COMAet occurs. However, the histopathology of surgical speci- al. (unpublished data) in the endemic zone of the ort- mens, or laparoscopic biopsies, has been reported by hern Altiplano of Bolivia, infected Aymara children un- many investigators from different countries. In contrast, doubtedly participate in the transmission of the disease the literature on the experimental pathology of fasciolia- in given localities owing to their defaecating habits. sis is extensive. A large review on pathology, sympto- Dietary habits of the human populations are very im- matology and clinical manifestations has been made by portant in fascioliasis. Watercress and other aquatic ve- CHE & MOlT(1990). getables able to carry attached metacercariae and inclu- Pathogenesis depends on the number of flukes that pe- ded in the human diet in different countries serve as netrate the intestine wall and invade the liver. In animals vehicles of the infection.The habits of eating raw water- the mortality rate is inversely proportional to the number cress and other vegetables cause the metacercariae to en- of flukes in the liver. The penetration of the intestinal ter the human alimentary tract, but the possibility of wall may cause focal haemorrhage and inflammation. being infected by means of drinking water carrying floa- The major pathological effects correspond to parasite ting metacercariae cannot be neglected (BARGUESet al., migration through the liver parenchyma for 4-6 weeks or 1996a). In some countries, such as in China, where vege- longer, flukes digesting hepatic tissue and causing exten- tables are always cooked for eating, infection may rarely sive parenchymal destruction with intensive haemorrha- occur by ingestion of unboiled drinking water, or from ges and inflammation. Migration tracks may be observed the metacercariae on cutting boards and other kitchen in histological sections. Migratory flukes sometimes die, utensils (CHEN & MOlT, 1990). leaving cavities filled with necrotic debris, and when The epidemiological analysis made by CHE & MOlT these heal, considerable areas of the liver may be repla- (1990) on the existing data shows that there is no marked ced by scar tissue (SMITHERS, 1982). In man, the pre- seasonal incidence, human infections occurring nearly sence of the parasites in the bile ducts causes fewer pat- throughout the year, that distribution by sex is very simi- hogenic effects, although inflammation resulting in lar, although in Egypt a higher prevalence was observed fibrosis, thickness and expansion is common (CHE & in women (10,3%) than in men (4,4%)(FARAG et al., MOlT, 1990). ISSEROFF,SAWMA& REI 0 (1977) sug- 1979), and that all age groups can be affected. However, gested that the extensive hyperplasia resulting in enlar- studies carried out on the Bolivian Northern Altiplano gement of the bile ducts is mediated by proline synthesi- endemic zone have demonstrated that children between zed and released by the parasites. Both in animal and 5 and 15 constitute the most infected age group (ESTE- human infections, anaemia is one of the most characte- BANet al., 1997a, b). Fascioliasis is predominantly a ru- ristic symptoms, especially in heavier infections (Bo- 178 S. MAS-COMA & M.D. BARGUES

RAY, 1969; DAWES &HUGHES, 1970). Blood loss into glandular epithelial hyperplasia. All layers of the wall the bile seems most probably to be an important, if not contain patchy infiltrates with Iymphocytes, plasma cells the only, factor contributing to severe anaemia. and eosinophils. Lithiasis, often multiple, in the common Immunologically, cell- and/or antibody-mediated res- bile ducts and gall bladders is very common (CHEN & ponse varies from host to host, and in the same host, ac- MOTT, 1990). cording to the phase of the infection (OLDHAM, 1985). The microscopic changes may be specific or non-spe- Immunity to reinfection differs greatly from host to host. cific. Generally, the migration tracks can be found in the Disease is self-limiting in cattle, as well as in rats, gui- liver and other organs. The walls of the tracks in the liver nea pigs and rabbits, resistance being acquired during often contain Charcot-Leyden crystals and eosinophils. the primary infections, but severe hepatic lesions and The cavities of the tracks are filled with necrotic cellular high mortality may occur, particularly in young or debi- debris, including hepatocytes, fibrin and red cells. A litated animals. In sheep, as in goats, hamsters and mice, considerable eosinophilic infiltrate surrounds the tracks. low or no resistance is seen and the infection is highly Longer tracks can cross several hepatic lobules. In older pathogenic in both the acute and chronic phases, death lesions macrophages, Iymphocytes, eosinophils and fi- being a usual sequela in heavy infections (BORAY, 1969; brous tissue are observed. Focal calcification is someti- SMITHERS,1982). In man, studies on immunity are limi- mes seen in the margin of the necrotic debris. Calcifica- ted, although it is generally believed that man is not a tions may form the outline of a dead fluke (CHEN & suitable host, most migrating flukes becoming trapped in MOTT,1990). the liver parenchyma and dying without reaching the Egg granulomas have been described (ACOSTA-FE- bile ducts (ACOSTA-FERREIRA, VERCELLl-RETTA & RREIRA, VERCELLI-RETTA& FALCON], 1979; GOOD- FALCONI,1979). Considerable tissue reaction and calci- MAN, HENDERSON & CULLlTY, 1973; IONES et al., fication of the bile passages due to the flukes have been 1977). The portal triads were dilated and oedematous recorded (ACOSTA-FERREIRAV, ERCELLl-RETTA& FAL- with infiltrates of lymphocytes and eosinophils. Bile CONI, 1979) and a spontaneous cure of the infection is duct proliferation, periductal fibrosis, necrotizing arterial not uncommon (BORAY, 1969). vasculitis and portal venous thrombosis were frequent. Disease is chiefly confined to the liver, so that the Immature flukes may deviate during migration, enter most important pathogenic sequelae are hepatic lesions other organs and cause ectopic fascioliasis. In cattle, F. and fibrosis, and chronic inflammation of the bile ducts. hepatica is frequently observed in the lungs (SOULSBY, Worth mentioning is that, unlike clonorchiasis or opist- 1965). In man, the most frequent ectopic lesions are horchiasis. there have been no reported associations with those of the gastrointestinal tract (ACOSTA-FERREIRA, biliary carcinoma. VERCELLl-RETTA & FALCONI, 1979; PARCK et al., According to CHEN & MOTT (1990), human liver is 1984). Other ectopic lesions are in: abdominal wall (To- usually enlarged with a smooth or uneven surface. The TEV & GEORGIEV, 1979), pancreas (CHITCHANG,MI- most common macroscopic lesions are multiple soft, ye- TAR UM & RATANANIKOM,1982), spleen (WEI, 1984), llowish or grey white nodules ranging 2-30 mm in dia- subcutaneous tissue (AGUIRRE ERRASTIet al., 1981b; meter, which microscopically appear to be eosinophilic GARCIA-RODRIGUEZet al., 1985; PARCKet al., 1984), abscesses. Haemorrhagic stippling appears at the margin heart (CHO et al., 1994), blood vessels, the lung and of the nodules. White or yellow striae are observed on pleural cavity (GARCIA-RODRIGUEZet al., 1985; PARCK the liver capsule. Close to the nodules. ribbed or vermi- et al., 1984), brain (RUGGIERI, CORREA & MARINEZ, form formations with similar colour and consistency as 1967), orbit (GARCIA-RODRIGUEZet al., 1985; CHO et nodules are also observed. Hepatic capsular thickening al., 1994), skeletal muscle, appendix (PARCK et al., of varying degree appears, and in a few cases the entire 1984) and epididymis (AGUIRREERRASTIet al., 1981 b). hepatic capsule is thickened. Subcapsular lymphatic ves- Such ectopic flukes never achieve maturity. The usual sels are dilated. The lymph-nodes near the porta hepatis pathological effects of ectopic lesions are due to the mi- may be markedly enlarged. In cases with marked invol- gratory tracks causing tissue damage with inflammation vement of the peritoneal wall and the liver surfaces, ye- and fibrosis. Parasites may be calcified or become incor- How and opalescent ascites was present. Apart from mild porated in a granuloma (FACEY& MARSDEN,1960). splenomegaly in 2 patients, no significant portal hyper- From the clinical point of view, the following periods tension was found in 18 cases by Japaroscopic examina- can be distinguished (FACEY& MARSDEN, 1960; MAN- tions (MORETO& BARRON, 1980). GOS & MENZIES, 1973): the incubation period (from the The common bile ducts are usually large and dilated ingestion of metacercariae to the appearance of the first and the wall is thickened on palpation. The gall bladder symptoms), the invasive or acute phase (corresponding wall is greatly thickened and oedematous. Multiple, gre- to fluke migration up to the bile ducts), the latent phase yish-white subserous nodules are present and adhesions (beginning with the maturation of the parasites and star- of the gall bladder to adjacent structures are common. ting of oviposition), and finally the obstructive or chro- The mucosal folds of the gall bladder are prominent. The nic phase. wall of the gall bladder appears thickened owing to mus- The incubation period varies considerably depending cular hypertrophy and perimuscular fibrosis. There is on the number of metacercariae ingested and the host's Human liver flukes: a review 179 response. The period of incubation in man has not yet cholecystitis and cholelithiasis of origins other than F. been accurately determined: only «a few» days (RAGAB hepatica infection. Hepatic enlargement may be associa- & FARAG, 1978), 6 weeks (RIMBAULT, 1981), or 2-3 ted with an enlarged spleen or ascites. If obstruction is months (HARDMA, laNES & DAVIES, 1970). present, the gall bladder is usually enlarged and oedema- In the acute phase, the symptomatology is due mainly tous with thickening of the wall. Lithiasis of the bile duct to the mechanical destruction of the liver tissue and of or the gall bladder is frequent. Stones are usually small the abdominal peritoneum by the migrating larvae cau- and multiple. The bile duct and the gall bladder may sing localized or generalized toxic and allergic reactions contain blood mixed with bile (haemobilia), blood clots lasting 2-4 months. However, in endemic areas, the in- and fibrinous plugs.The diagnosis has usually been con- fection with F. hepatica is usually repetitive and the firmed at laparotomy by the finding of flukes in the com- acute lesions are superimposed on chronic disease. Thus, mon bile duct or in the gall bladder, commonly associa- the acute phase may be prolonged and overlap on to a la- ted with cholangitis and cholelithiasis. With proper tent or an obstructive phase. The major symptoms of this clinical management - removal of the obstruction and phase are fever, abdominal pain, gastrointestinal distur- temporary biliary drainage - the prognosis is good bances and urticaria. Fever is usually the first symptom, (CHEN& MaTT, 1990). usually low or moderate but sometimes reaching 40° C, The outstanding abnormal laboratory finding in all and in heavily infected cases as high as 42° C; it may be phases of F. hepatica infection is eosinophilia (always remittent, intermittent or irregular with higher tempera- greater than 5%, the highest detected being 83%), ac- ture in the evening. In some cases, a low, recurrent fever companied by leucocytosis (over 10000/mm3 up to lasted for a long time (4 to 18 months). Abdominal pain, 43000/mm3), especially in the acute phase. Anaemia is from mild to excruciating, was generalized at the outset common, but usually not very severe (mostly between but usually localized in the right hypochondrium or be- 7,0 and 11,0 g/dl haemoglobin; levels as low as 2.8 and low the xyphoid, sometimes vague. Among gastrointes- 4.0 g/dl have been reported). The erythrocyte edimenta- tinal disturbances, loss of appetite, abdominal flatulence, tion rate (ESR) may be high in the acute phase (possibly nausea and diarrhoea are common, whereas vomiting reaching 165 mm in an hour). Abnormal liver function and constipation are infrequent. Urticaria, with derrnato- tests may be seen both in the acute and in the obstructive graphia, is a distinctive feature in the early stage of the phases, but high serum bilirubin levels are associated fluke invasion and may be accompanied with bouts of with the obstructive phase (CHEN& MaTT, 1990). bronchial asthma. Among respiratory symptoms, non- Serum immunoglobulin studies (laNES et al., 1977; productive cough is common, whereas chest pain occurs SALEM, ABOUBASHA& FARAG, 1987; SAMPAIOSILVA occasionally. In the acute phase, the following signs may et al., 1985) have shown that levels for IgG, IgM and appear on physical examination: hepatomegaly and sple- IgE are usually elevated. Specific IgE antibodies were nomegaly, ascites, anaemia, chest signs and jaundice detected in 48% of the patients. Total and specific IgE (CHE & MaTT, 1990). levels have been shown to be positively correlated with The latent phase can last for months or years. The pro- the egg burden, age, clinical features and degree of eosi- portion of asymptomatic persons in this phase is unk- nophilia. Ig A levels are usually normal. nown. Diagnosis of infection may be confirmed after cli- In man, complications may comprise bleeding and bi- nical suspicion or in epidemiological surveys by finding liary cirrhosis, which may be the major causes of death. the eggs in the duodenal fluid and/or in the stool. An Death is rare as the infection is usually sporadic and the unexplained, prominent eosinophilia may suggest a hel- overall prevalence is low. Only 8 deaths related to fas- minthic infection. These persons may have gastrointesti- cioliasis have been reported in the recent literature nal complaints or one or more relapses of the acute (CHEN& MaTT, 1990). symptoms during this phase (FACEY& MARSDEN,1960; MANGOS& MEZIES, 1973). Diagnosis A chronic or obstructive phase may develop after months to years of infection. Adult flukes in the bile Coprological examination is still the main method for ducts cause inflammation and hyperplasia of the epithe- diagnosis.However, serological methods have been de- lium. Thickening and dilatation of the ducts and the gall veloped and have confirmed the diagnosis in the acute bladder walls ensue. The resulting cholangitis and cho- phase of the disease, in which no eggs can be found in lecystitis, combined with the large body of the flukes, stools owing to the absence of sexually mature adults in are sufficient to cause mechanical obstruction of the bi- this initial phase. They are also useful for monitoring liary duct, which is comparatively small in diameter. post-treatment evolution. The proportion of those whose infection develops into Parasitological diagnosis is based on egg identification the obstructive phase or their prognosis has not been de- in the stool or in duodenal or biliary drainage. The possi- fined.The clinical manifestations in this phase, such as bility of a spurious infection must always be taken into biliary colic, epigastric pain, fatty food intolerance, nau- account, the presence of eggs in faeces being only the sea, jaundice, pruritus, right upper-quadrant abdominal consequence of the consumption of infected liver from tenderness, etc., are indistinguishable from cholangitis, ruminants. To avoid false fascioliasis, stool examination 180 S. MAs-CoMA& M.D. BARGUES should be repeated after a few days of a liver-free diet. enzyme-linked immuno-electrotransfer blot (EITB), Fal- Methods for the detection of the presence of parasite con" assay screening test-enzyme linked irnrnunosor- adults can also be applied.Adult flukes and/or eggs may bent assay (FAST-ELISA), automated assay of anti-PI be found in the biliary tract or in the bile at exploratory antibodies, circulating antigen, and circulating immune laparotomy. Histological examination of liver biopsy complex (CIC). Almost all the serological tests are material may occasionally reveal an egg granuloma or highly sensitive. With partially purified somatic or ex- sections of the fluke. It must also be taken into conside- cretory-secretory products of adult F. hepatica as anti- ration that the prepatent period is about 3-4 months, so gen, ELISA (ESPINOet al., 1987; HlLLYER, 1981; HILL- that coprological techniques become useful only after YER & SANTIAGODE WElL, 1979; LEVlNE, HILLYER& that moment. FLORES, 1980), IFA(BULAJICEet al., 1977; CAPRONet According to CHEN & MOTT (1990), coprological al., 1973) and CEP (HILLYER, 1981; HILLYER & SA- techniques, ranging from a simple direct smear to diffe- TIAGODE WElL, 1981) have been reported to have the rent concentration methods, have been used. Egg con- highest sensitivity and specificity. IFA was reported to centration has been achieved by flotation, sedimentation have 92-96% sensitivity in the acute phase of the infec- (ASHTON et al., 1970; BENDEZU, FRAME & HILLYER, tion by CAPRONet al. (1973). In chronic infection with 1982; BOLBOL, 1985; BORAY, 1969; DE LEON, QUINO- F. hepatica, IFA and CEP may not be positive (CAPRON NES& HILLYER, 1981; FARAGet al., 1979; KNOBLOCH et al., 1973). However, cross-reactions in other helmint- et al., 1985; STORKet al., 1973) and the cellophane fae- hic infections such as , and fi- cal thick-smear techniques (Kato, Kato-Katz) (KREMER lariasis have been reported. Crude F. hepatica antigen & MOLET, 1975). The sedimentation technique is more may have cross-reactivity with other trematodes (HILL- accurate and sensitive than flotation techniques as most YER, 1981). The specificity of the serological tests may of the hyperosmotic solutions distort the eggs (BORAY, be improved by elimination of cross-reactivity with anti- 1969). The Kato cellophane faecal thick-smear techni- bodies to Schistosoma and other trematodes through par- que has the advantages of being rapid, having low cost, tial purification of the antigen (HILLYER & SANTIAGO being reproducible and quantitative. The Kato technique DE WElL, 1979). Summing up, no consensus as to the has been used in the diagnosis of experimental F. hepa- optimal antigen or test system has been reached so far tica infection (LEVlNE, HILLYER & FLORES, 1980) but, (STORKet al., 1973; CHEN& MOTT, 1990). although it may be useful in epidemiological studies, its Worth mentioning are several experimental studies ha- relatively low sensitivity limits its clinical application. ving shown that after effective chemotherapy, anti-F. he- According to KNOBLOCHet al. (1985), rapid sedimen- patica antibodies became undetectable: ELISA titres tation (using 20 g faeces on each of 3 consecutive days), dropped rapidly and the test was useful for the evalua- although inconvenient, seemed to be largely more sensi- tion of chemotherapeutic success (HILLYER & SAN- tive than the merthiolate-iodine-formaldehyde concen- TIAGODE WElL, 1979), precipitins in CEP test disappea- tration method (MIFC)(using 1 g of faeces in a single red by 4 weeks (HlLLYER& SANTIAGODE WElL, 1981), examination) or the Enterotest (single examination of and CEP and ELISA became negative 3-4 weeks later duodenal fluid). Among five concentration techniques (LEVlNE, HILLYER& FLORES, 1980). However, double compared by AKAHANEet al. (1975), the recovery rates diffusion and IHA became negative after a longer period were: formalin-ether method, 5,3%; HCl- ether method, (1-2 years after treatment) according to GARCIA-RoDRI- 7,8%; Weller-Damrnins modification method, 37,7%; GUEZ,MARTINSANCHEZ& GARCIALUlS(1985). citrate buffer-Tween 80-ether method, 25,3%; and The clinical presentation may be helpful for the diag- AMS III (Tween 80) method, 30,5%. nosis (CHEN & MOTT, 1990). Fascioliasis is frequently The early diagnosis of the acute phase may be achie- considered among the differential diagnoses in a well- ved by immunological techniques. known endemic area. However, in areas where the dise- Skin tests employing an antigen prepared from the ase is rarely reported or absent, physicians may not con- adult flukes (SMITHERS,1982) or purified fraction of F. sider this diagnostic possibility. History of ingestion of hepatica (STORK et al., 1973) have been used occasio- raw wild or cultivated watercress or other vegetables, or nally since the early 1960s. The tests were simple and other contaminated food or water may be suggestive of sufficiently sensitive to propose a diagnosis of the infec- the infection. tion (CAPRONet al., 1973) but not very specific (STORK According to ARJONAet al. (1995), the clinical situa- et al., 1973). The technique is rarely used nowadays. tions in which the diagnosis of F. hepatica infection During the past 2 decades, with the development of should be considered are: history of watercress inges- new technology, different serological tests have been tion, eosinophilia, fever of unknown origin, atypical ab- used both in experimental infections and in humans (see dominal pain, focal intrahepatic lesions, granulomatous review by CHEN & MOTT, 1990). These include: com- hepatitis, serositis and meningitis with peripheral or plement fixation (CF), immunofluorescence assay (IFA), fluid eosinophilia, family history of , biliary counter-electrophoresis (CEP), enzyme-linked imrnuno- colic or cholangitis, and normal ultrasonography. Eosi- sorbent assay (ELISA), kinetic-dependent ELISA, dou- nophilia has also been successfully used for a first selec- ble diffusion, indirect haemagglutination (IHA), tion in general surveys (GlL-BENlTO et al., 1991). Human liver flukes: a review 181

In the acute phase the clinical presentation includes fe- Hexachloro-para-xylol has been effectively used at a ver, pain in the right hypochondrium, prominent eosi- dose of 50-80 mg/kg body weight daily divided into 3 nophilia with leucocytosis, anaemia and a moderately to doses given orally for 7 consecutive days in China (SUN, significantly high ESR. CHAI & CHENG, 1984; WANG et al., 1981) and at a dose In the chronic (latent and obstructive) phase the clinical of 60 mg/kg daily for 5 days in the former USSR (KHAS- picture is attenuated and easily confused with other disea- HIMOV & KAMARDINOV, 1975; RAKHMANOV,1987), ses. The classic pattern includes: vague gastrointestinal side effects including gastrointestinal complaints and complaints, pain in the right hypochondrium or epigas- dizziness. trium, cholecystitis, cholangitis and bile duct or gall blad- Bithionol (Bitin) is used at a dose of 50 mg/kg daily, der stones. The liver is usually enlarged with or without divided into 3 oral doses on alternate days for 15 days. In pain on palpation. Ascites may appear in advanced cases. cases of fascioliasis resistant to emetine and praziquantel Non-invasive diagnostic techniques which can be used treatment, bithionol achieved cure in dosages of 50 for the diagnosis in this chronic phase are radiology, ra- mg/kg daily for 10 alternate days (GRADOS& BERRO- dioisotope scanning, ultrasound and computed tomo- CAL, 1977) or 40 mg/kg daily for 15 alternate days graphy (CT). Fascioliasis has been diagnosed by: abdo- (BHATTACHARYYA,1985). The side effects are mild and minal and chest X-ray examination; oral, percutaneous are related to the gastrointestinal tract including anore- and intravenous cholangiography; and endoscopic retro- xia, nausea, vomiting and abdominal pain. grade cholangio-panchreatography (ERCP). However, Daily oral doses of 1,5 g of metronidazole for 13, 14, the findings are not pathognomonic of F. hepatica infec- 21 and 28 days were effective in 4 patients (NIK-AKH- tion. Radioisotope liver scan may be useful in the diag- TAR& TABIBI, 1977), but a smaller total dose of 4 g was nosis of fascioliasis, patterns observed being however reported to have failed to cure a chronic infection (ECK- not specific. Ultrasound has proved useful in the diagno- HARDT& HECKERS,1981). sis of the pathological lesions secondary to F. hepatica Albendazole is a broad-spectrum which infection in the liver and the biliary tract (BASSILY et al., has been demonstrated to be effective in cattle at a single 1989). In fascioliasis, the ultrasound image is usually oral dose of 15 mg/kg body weight (MI et al., 1983) normal and the individual adult flukes are not visualized and sheep at a single dose ranging from 3.8 to 7.5 mg/kg by the current ultrasound technology. CT has a high le- body weight, but the efficacy of the drug against the im- vel of resolution for auxiliary and pathological diagnosis mature flukes was lower (JOHNS& DICKESON,1979). of F. hepatica infection. Several months after treatment Mebendazole, in a daily dose of 4 g for 3 weeks, was for F. hepatica infection, a marked improvement in the reported to have cured a F. hepatica infection diagnosed CT images has been shown by different investigators. clinically and serologically in the invasive phase (DU- The multiple hypodense areas in the liver were reduced GERNIERet al., 1986).Diamphenetide was effective both significantly in number and size (DEMIGUEL et al., in vitro (FAIRWEATHER,A DERSO & THREADGOLD, 1984; GOEBEL,MARKWALDER& SIEGENTHALER,1984; 1988) and in experimentally infected sheep at 120 mg/kg PAGOLASERRANOet al., 1987; TAKEYAMAet al., 1986). body weight (JIN et al., 1984). Rafoxanide was reported CT scan can be a useful tool for the diagnosis of the di- to be effective in sheep and cattle (CAI, 1988) and was sease and its possible complications as well as followup used in the treatment of a child with fascioliasis (YUR- of the patient's response to the treatment. DAKOK,1985). In both the acute and chronic infections, ectopic locali- Niclofolan, widely and successfully used for veterinary zation of the parasite may cause a confusing clinical pre- purposes in China (CHEN & MOTT, 1990), has been ap- sentation. plied twice for human treatment showing such a toxicity An acute dysphagia and laryngeal obstruction after in- that clinical use cannot be recommended (ECKHARDT& gestion of raw liver of sheep or goats, was formerly con- HECKERS,1981; RESHEF,LOK & SHERLOCK,1982). sidered to be due to invasion of immature F. hepatica Worth mentioning is that Fasciola may be the only ge- and was known as «Halzoun» or pharyngeal fascioliasis nus of trematode that has practically no response to pra- (FACEY& MARSDE , 1960). Now it is attributed to the ziquantel, most clinical reports having shown that prazi- ingestion of nymphs of Linguatula serrata, a pentasto- quantel failed to cure F. hepatica infections, even at high mid parasite (CHATTERJEE,1975). doses (CHEN & MOTT, 1990). In vitro and in mice, rats and sheep, F. hepatica is refractory to praziquantel (AN- DREWSet al., 1983; PEARSON& GUERRANT,1983). Treatment is effectively used in veterinary medi- Emetine and dehydroemetine are the classic drugs and cine against both adult and immature F. hepatica. In ex- have been used widely, at a usual dose of 1 mg/kg daily perimental studies in sheep, BORAY et al. (1983) and for 10 days given intramusculary or subcutaneously TURNER et al. (TURNER, ARMOUR& RICHARDS,1984) (CHEN & MOTT, 1990). They are effective and are still showed that doses of 2,5-5,0 mg/kg body weight elimi- being used. However, owing to their side effects, several nate almost all the flukes (98,1-100% reductions) 12 we- other types of drugs have been developed during the past eks after infection.A higher dose of 10 mg/kg body decade. weight achieved reductions of 93-98% of the flukes, one 182 S. MAS-COMA & M.D. BARGUES week after infection (BORAY et al., 1983). Concerning the livestock farming community. Forecasts of outbreaks humans, there are some preliminary clinical data already may be made based on climatological data and epide- published, although this drug is not yet registered for hu- miological models. Recommendations for control mea- man use (CHEN& MOTT, 1990). Three patients were ef- sures should be made on a preventive rather than a cura- fectively treated (12 mg/kg single dose for the first pa- tive basis, and all measures have to be considered from tient; 5 mg/kg first dose, followed by 10 mg/kg second the point of view of the economy and assessment of lo- dose on the next day for the second patient; 10 mg/kg cal topographical and meteorological conditions. The ef- single dose for the third patient). Clinical tolerability ficiency of fascioliasis control depends on the correct was excellent in 1 of these patients while in the other 2 and integrated application of: A) reduction of the para- patients after a single dose a transient febrile episode site load of the animal hosts and pasture contamination with reversible liver function alteration was observed by regular strategic use of drugs (preventive treatment in (MARKWALDERet al., 1988; WESSELY, REISCHlNG & appropriate year periods according to different regions); HEINERMANN,1987; WESSELY et al., 1988). Four pa- B) reduction of the number of snails by physical, chemi- tients, whose diagnosis was confirmed by specific im- cal and biological means; C) reduction of the risks of in- munoelectrophoresis and indirect haemagglutination, fection through correct farm management practices (ro- were also successfully treated with a single dose of 10 tational system through fluke-infected and fluke-free mg/kg (LE BRASet al., 1989). Three other patients have paddocks, combined with effective treatment) (BORAY, been treated with a single oral dose of 10 mg/kg body 1982).A wide variety of candidates for vaccination has weight; two of them recovered steadily over 3 weeks, been proposed and studied at veterinary level, and the but the third again felt ill after a month of slight impro- possibility of disposing of an effective vaccine for rumi- vement, very few eggs being found in subsequent stool nants seems feasible even for the near future, but further examinations, so that a second treatment of triclabenda- work is needed. zole in 2 successive postprandial doses (10 mg/kg) 12 Contrarily to the veterinary aspect, owing to the well hours apart were given (LOUiAN et al., 1989). known important impact of fascioliasis on production No new drugs have been developed during the last 15 (DARGlE, 1986), intermediate snail host control has un- years for the treatment of fascioliasis and drug resis- fortunately not received the attention from public health tance in F. hepatica has already been reported to affect officials required to definitively eliminate transmission the efficacy of the drugs against immature stages. Drug (CHEN & MOTT, 1990). Intensive agricultural methods combinations have been recently tested and it was must be applied to reduce suitable snall habitats. Besides shown that their synergistic action increased efficacy physical methods, there are available control strategies against immature flukes, removed resistant flukes and which consist of the use of chemical molluscicides, natu- would also reduce the development of resistance (Bo- ral molluscicides of plant origin, biological control (in- RAY,1994). cluding predators, competitors, the decoy effect and rela- Among the current drugs, until triclabendazole is re- ted phenomena, parasitic castration, interspecific gistered for human use, bithionol seem to be the drug of trematode antagonism, and pathogens), genetic manipu- choice, although its treatment course is comparatively lation, and engineering control (MALEK, 1985; COMBES long. Emetine and dehydroemetine are still effective & CHENG, 1986). The practical application of chemical drugs. Triclabendazole, albendazole and niclofolan are methods in the control of snails is of doubtful value, re- quite effective in veterinary infections, but the absence quires labor and equipment, and regular yearly strategic of toxicological information required for registration or moIluscicide applications (BORAY, 1982). clinical trials in man, their use cannot yet be recommen- ded (CHEN& MOTT, 1990). FASCIOLA GIGANTICA Prevention and control Morphology The prevention of human fascioliasis may be achieved simply by strict control of watercress and other metacer- First described from a giraffe (COBBOLD, 1855), the cariae-carrying aquatic plants for human consumption, giant liver fluke has a morphology similar to that of F. especially in endemic zones. Commercial growing of hepatica, but is much larger and slightly narrower, mea- watercress should be carried out under completely con- suring 24-76/5-13 mm. The average length/width ratio is trolled conditions, without access for snails and rumi- 4,39-5,20 in F. gigantica, while it is 1,88-2,32 in F. he- nants. Worth mentioning in endemic areas is that the patica (SAHBA et al., 1972). Other differental characte- community should be appropriately informed about the ristics are the following: in F. gigantica, the shoulders disease, its transmission and its danger. are less developed, the cephalic cone is shorter, and the As for control measures, previous epidemiological stu- caeca are more branched than in F. hepatica, especially dies may provide for general recommendations on the those toward the midline of the body (the centipedal appropriate time for treatment with effective drugs to branches). The branches of the ovary are longer and achieve economic control, and better information from more numerous in F. gigantica and are smaller and club- Human liver flukes: a review 183 shaped in F. hepatica. The average distance between the In Africa, human infection with F. gigantica has been posterior border of the body and the posterior testis is recorded in many countries. Human cases have been longer in F. glgantica (14,9 mm; range: 6-19 mm) than diagnosed in Rwanda and Burundi (JANSSENSet al., in F. hepatica (7,78 mm; range: 3-13 mm)(SAHBAet aI., 1968; HAMMOND, 1974), Malawi (SPECKHART,1969; 1972). The eggs are morphologically similar to those of HAMMOND, 1974), Rhodesia (PERRY, GOLDSMID & F. hepatica but larger, measuring 150-196/90-100 urn. GELFAND, 1972; HAMMOND, 1974), Uganda (HAM- VARMA(1953) erected F. indica for the liver flukes of MOND,1974), southern Africa (GELFAND,1971; GOLDS- India, thus differentiating it from F. gigantica. Howe- MID, 1975), Zaire (FAIN, DELVILLE & JACQUERYE, ver, SARWAR (1957) and KENDALL& PARFITT (1959) 1973), Cameroon and Gabon (GRANGE et al., 1974; considered both identical. As already stressed by KEN- HAMMOND,1974), Madagascar (MOREAUet al., 1975), DALL (1965), morphological differences detected seem Zambia (HIRA, 1976), Egypt (MANSOURet aI., 1983), to be attributable to differences in fixation and moun- Mali (MAIGA et al., 1991), and Cabo Verde archipelago ting of materials, normal biological variation within a (CRUZ E SrLvA et aI., 1972). species or even to the use of material from different In Asia, reports of human fascioliasis sometimes do hosts. At any rate, evidence from experimental infec- not refer to the species of Fasciola because both F. he- tions suggests that various strains of the parasites have patica and F. gigantica occur in the country in question developed because of geographic isolation (MANGO, or because the specific identity is not agreed upon; MANGO &ESAMOL, 1972; SRIVASTAVA & SINGH, among these countries are Iran, China, Korea, and Japan. 1974; KHAJURIA&BAll, 1987). In Iran, FARID(1971) identified three cases by stool exa- Mapping of rDNA genes did not allow BLAIR & Mc mination and cited 11 other cases previously reported in MANUS (1989) to detect intraspecific variation in F. gi- that country. In Korea, a case was described by CHO et gantica from Indonesia and Malaysia. Worth mentioning al. (1976) in which a complete worm, identified as Fas- is that one specimen of Fasciola sp. from Japan (where ciola sp., was removed during bile duct exploration; the both F. hepatica and F. gigantica overlap, causing identi- morphology of this worm was considered intermediate fication problems because of the detection of intermediate between F. hepatica and F. gigantica. This is also the forms) yielded a restriction map identical to that of F. gi- view of Japanese investigators concerning the Japanese gantica. Studies by ADLARDet at. (1993) on the nucleo- Fasciola (WATANABE(1965). In Japan, only 19 cases tide sequence of the 3' end of the second internal transcri- were reported up to 1974, the last of which was a 49- bed spacer region (lTS-2) of the ribosomal DNA showed year-old woman from Kyoto City; the parasite was iden- sequence divergence between F. hepatica and F. gigan- tified as Fasciola sp. on the basis of eggs in the faeces tica (2,8%) and the sequence of Fasciola sp. from Japan and bile (YOSHLDAet al., 1974). Sometimes, however, closely matched that of F. gigantica. More recently, HAS- human infection was diagnosed as concretely caused by HIMOTOet al. (1997) concluded that Japanese Fasciola sp. F. gigantica (ISHIGAMIet al., 1973). Human parasitation must be considered a strain of F. gigantica, taking into ac- by F. gigantica has also been recorded in former USSR count the absence of differences in the sequences of the (Tashkent and Uzbekistan), Vietnam, and Iraq (HAM- ITS-2 and the similarity of the sequences of the mitochon- MOND, 1974). Recently, SADYKOV(1988), in post-mor- drial cytochrome c oxidase subunit I (COl). tem examinations, detected fascioliasis in 81 inhabitants of the Samarkand region, USSR, in 1968-1986; F. hepa- tica was in 45, F. glgantica in 25, and both species in 11; Location and definitive hosts deaths were not due to fascioliasis, which was detected It is a common parasite of the bile ducts and gall blad- incidentally, suggesting that it is much more frequent der of domestic and wild herbivorous animals, especially than reported. More recently, TESANA, PAMARAPA& ruminants, in Africa and Asia. Reported definitive host SIO(1989) reported human fascioliasis by F. gigantica species are sheep, goat, cattle, and buffalo, camel, pig, from Northeast Thailand. horse, donkey, larger antelope, deer, giraffe, and zebra. Cases have also been reported from humans in Hawaii Occasionally F. gigantica has also been reported in nu- (ALlCATA, 1938, 1953). ALlCATA& BONNET(1956) sta- tria and in monkeys.Several species of laboratory ro- ted that at least 19 outbreaks occurred on the island up to dents can also be infected experimentally (BORAY, 1956. 1982). Many other African wild animals have been also As already stated by HAMMOND(1974), human infec- found naturally infected (Losos, 1986). tion with F. gigantica may be more common than is thought, but has not been reported more frequently be- cause it has not been looked for adequately. A further Reports in humans possible reason for the apparently low human infection There are relatively few records of human infection rate is that the disease may be mild and cause few symp- with F. gigantica, and furthermore, nearly all of these re- toms in persons of given ethnic groups. Thus, the symp- fer to single cases or to very small numbers of patients, tomatology observed in indigenous persons in Malawi although this parasite is widespread in many areas of the were mild as compared with those in three Europeans in tropics and subtropics. central Africa and in some cases reported in Hawaii. At 184 S. MAS-COMA & M.D. BARGUES any rate, it is impossible to compare these reports in de- North America, where the snail intermediate hosts are tail without information on the level of infection. suitable for both species. Generally, in tropical countries GOLDSMID(1975) believes that the overall paucity of where both species exist together, F. gigantica is usually human records of fascioliasis in the wetter areas of sout- endemic in the lower regions while F. hepatica is ende- hern Africa, even where fascioliasis in domestic stock is mic in the highlands (BORAY, 1982). common, may be due to the fact that fascioliasis, being a rural infection, is often not diagnosed in such country Life cycle clinics which have no laboratory facilities. Probably this is also the case in other parts of Africa and other parts of The life cycle is essentially similar to that of F. hepa- the world. Even where laboratory facilities are available, tica and includes Iymnaeid snails, especially those spe- infected patients do not always pass eggs, as in the case cies associated with standing or slow-flowing water con- recorded by PERRY,GOLDSMID& GELFAND(1972). taining abundant vegetation, in which sporocyst and rediae develop (ALICATA, 1938, 1953; THAPAR&TAN- DON, 1952; DINNIK & DINNIK, 1956, 1963; KE DALL, Geographical distribution 1965). The miracidium of F. gigantica shows a different According to BORAY(1982), F. gigantica is present in behaviour from that of F. hepatica. The miracidium of large areas of the African continent, from the Nile Delta F. hepatica is positively phototropic and negatively geo- in the north to the Cape Provinces of South Africa in the tropic, as an adaptation to the amphibious or water sur- south, including Sudan (MALEK, 1959), Senegal (VASST- face snails. In F. gigantica, it moves into deeper waters LIADES,1974), Chad (GRABER& OUMATIE,1964), Gui- which harbour Lymnaea natalensis in Africa and varie- nea-Bissau (MANDINGA, 1986), Ghana (ODEI, 1966), ties of L. auricularia in Asia. AUCATA (1938) observed Togo (GNINOFOU,1988), Niger (TAGAR-KAGAN,1977, 2 redial generations, only the second being cercarioge- 1979), Central African Republic (GRABER & THALL, nous. DLNNIK& DINNTK(1963, 1964) observed 3 redial 1979), Tanzania (MEGARD, 1975) and Kenya (WAMAE generations, cercariae alternating with rediae in all 3 ge- & CHERUIYOT,1990), as well as Mocambique, Ethiopia, nerations depending on environmental conditions. Ivory Coast, Liberia, Sierra Leone, Nigeria, Zimbabwe OGAMBO-ONGOMA& GOODMAN(1976) found 4 gene- and Angola (SCHILLHORNVAN VEEN, 1980; Losos, rations of rediae, the third and fourth being cercarioge- 1986), besides the already mentioned Rwanda and Bu- nous. Shedding of cercariae by the snail shows a noctur- rundi, Malawi, Rhodesia, Uganda, southern Africa, nal periodicity, emergence taking place in the dark phase Zaire, Cameroon, Gabon, Zambia, Egypt and Mali. It is (PRASAD, 1992). Shed cercariae attach to water plants also present in the islands of Cabo Verde (CRUZ E SILVA and become encysted metacercariae, which infect the de- et al., 1972), Zanzibar and Madagascar (BORAY, 1982). finitive host by ingestion. The intraorganic migration of The major endemic areas of F. gigantica are tropical the fluke up to the final location of the adult stage is si- Asia, Southeast Asia, and the Pacific regions, including milar to that of F. hepatica. countries such as old USSR (Tashkent, Uzbekistan, Turkmenia, Samarkand region), Iran, Iraq, China, Korea, First intermediate hosts Japan, India, Pakistan, Vietnam, Thailand, Laos, and fi- nally, in the Pacific, Malaysia, Philippines and Hawaii. Principal or obligatory intermediate snail hosts mentio- F. gigantica infection is one of the most important disea- ned for F. gigantica are: Lymnaea natalensis in Africa, L. ses threatening the livestock populations of India, Pakis- auricularia sspp. in the Near East, Middle East, Far East tan, Indonesia, Indochina, and the Philippines. Less im- and southern states of the old USSR, L. cubensis in North portant endemic areas of F. gigantica are the southern America on the gulf coast, L. rufescens in Asia and the parts of Europe, Turkey, the Near East, and some south- Indian subcontinent, L. rubiginosa in the Far East and ern states of the old USSR, particularly Armenia (Bo- Malaysia, L. swinhoei in South Eastern Asia, and the Phi- RAY,1982). lippines, and L. ollula in Hawaii and Japan. Alternate or In North America, according to PRICE (1953), there facultative host species are: L. truncatula in Africa, L. pe- seem to be 3 Fasciola types in animals in the USA: those regra in the Near East, Middle East, and southern states from various parts of the U.S. which are identical to F. of the old USSR, L. columella on the North American hepatica of the Old World, those from Texas and Flo- gulf coast, and L. viridis in the Far East (BORAY, 1982). rida, approaching F. gigantica, and those from the Gulf L. caillaudi in Egypt (FARAGet al., 1979), L. gedrosiana coast area which appear to be intermediate between F. in Iran, L. euphratica in Iraq and L. luteola in Nepal (Mo- gigantica and F. hepatica. This author stated two possi- REL& MAHATO, 1987), as well as L. bactriana, L. tenera bilities: whether they are a consequence of importations and L. subdisjuncta in Turkmenia (CHARVEY,1989) must into the Gulf coast area from India carried out in the ye- be added. ars 1875 and 1906, or simply the consequence of intras- These Iymnaeid intermediate hosts of F. gigantica are pecific variability of a same species. distinguishable from those of F. hepatica, both morpho- Overlapping infections may occur with both F. hepa- logically and as to habitat requirements. Their habitats tica and F. gigantica in some parts of Africa, Asia and are permanent water bodies rich in aquatic vegetation. Human liver flukes: a review 185

Interestingly, a planorbid species, Biomphalaria ale- source of metacercarial ingestion, the cane commonly xandrina, has recently been found naturally infected by being stripped by Africans with their teeth. F. gigantica larval stages in Egypt (FARAG & EL SA YAD, 1995). The epidemiological and transmission implica- Pathology, symptomatology tions of this finding remain unknown. and clinical manifestations In man, the disease is similar to that observed in F. he- Epidemiology patica infections. According to HAMMOND (1974), the Mode of spread, epidemiology and factors influencing pathogenicity varies and only mild and subclinical dise- the life cycle are similar for both F. hepatica and F. gi- ase with few symptoms sometimes occur in authochtho- gantica. There are, however, several differences: inter- nous persons. Signs and symptoms include severe epigas- mediate host snails are not drought-resistant as are mo- tric pain, fever and hypereosinophilia. Acute cholecystitis lluscan hosts of F. hepatica; the rate of development of was found by TESANA, PAMARAPA & SIO (1989). Worth the various stages of F. gigantica is slower; F. gigantica mentioning is the recovery of a worm from a painful sub- metacercariae survived longer at high temperatures and cutaneous swelling that moved from the right to the left are more susceptible to desiccation than F. hepatica me- side of the chest (FAIN, DELVILLE & JACQUERYE, 1973). tacercariae (BORA Y & ENIGK, 1964); Iymnaeid interme- Information on the pathology of the human liver infec- diate hosts need higher temperatures (>20° C) for their ted with F. gigantica was given by STEMMERMANN development (MEGARD, 1975). All this suggests that F. (1953a, b) from human cases in Hawaii. There were gigantica is a parasite which is adapted to tropical areas many soft, yellow elevations measuring 2-3 mm in dia- and to aquatic snails and to an aquatic environment for meter over the surface of both lobes. On sectioning the its transmission. This is related to the fact that F. hepa- liver, many pus-filled cavities from 3 to 2,5 mm in dia- tica prevails in temperate zones while F. glgantica is meter were noted in both lobes. The smaller cavities had predominant in tropical regions (OVER, 1982). soft, poorly defined, gray walls. The larger cavities had Prevalences of F. gigantica infection differ according ragged lining and brownish-red walls which varied from to different regions and zones, as well as to definitive I to 3 mm in thickness. The intervening parenchyma had host species and races. The following prevalences have a flabby consistency. The intrahepatic biliary ducts con- been recorded: 3% in Chad, 30% in the Central African taining the flukes were dilated. Microscopic examination Republic, 45% in Cameroon, 37% in Sudan, 30-90% in revealed both the smaller and the larger bile ducts to be Ethiopia, 16% in Uganda, 4-8% in Tanzania, 60-70% in filled with neutrophils, fibrin, and nuclear debris. The Zimbabwe, 50% in Ruanda and 33% in Kenya (ME- periportal connective tissue was extremely edematous GARD, 1975). and contained plasma cells, neutrophils, Iymphocytes, Human infection takes place by the accidental inges- and histiocytes. The ductal walls were greatly thickened. tion of raw vegetation, including watercress (ALlCATA & Large areas of the liver had undergone necrosis. Present Bo ET, 1956) containing encysyted metacercariae, within the necrotic areas were numerous Charcot-Ley- particularly in areas where infected cattle are permitted den crystals. The necrotic tissue was surrounded by vas- to roam (STEMMERMAN , 1953a, b). In Africa, human cular granulation tissue containing many neutrophils and infections with F. gigantica are believed to be caused by eosinophilic granulocytes, plasma cells, plump fibro- ingestion of watercress in Rwanda and Burundi (JANS- blasts, histiocyts, and giant cells. Some of these necrotic SENS et al., 1968), and infection could also occur after granulomatas spanned several hepatic lobules, but the chewing infested grass or green rice. Cercariae can also hepatic architecture of the uninvolved portions was well encyst on very small floating particles or on water sur- maintained. GRA GE et al . (1974) described hepatic cal- face, in which case they may be swallowed in contami- cification found surgically. nated drinking water. In Samarkand region, the old In animals, the disease also resembles that due to F. USSR, the relatively high human prevalence was related hepatica. Because of the larger size of this species and to the important percentage (10,5%) of green vegetables the slightly longer period of migration in the liver tis- sold in the Samarkand market, which presented encysted sues, the acute disease may be more severe in F. gigan- metacercariae (SADYKOV, 1988). GOLDSMID(1975) and tica infection. The chronic disease is similar to that cau- GELFAND (1971) believe that one of the reasons human sed by F. hepatica and deaths are caused by severe fascioliasis is rare in southern Africa may be the dietary anemia (BORA Y, 1982). Pathogenicity in cattle is similar habits of the Africans in this area, where water plants do to that of F. hepatica (SINCLAIR, 1967). SEWELL (1966) not seem to be an important source of food or relish and, found bilirubinemia and jaundice to be a marked feature in any case, are mostly eaten cooked. However, SPECK- of the terminal stages of subacute fascioliasis in cattle HART(1969), in Malawi, has pointed out that some vege- produced by F. gigantica. table plants are eaten uncooked, listing cabbage, tana- The normal definitive host of F. gigantica is cattle. F. posi, and mnadzi, and these may serve as sources of gigantica appears to be a parasite not well adapted to infection in swampy areas. This author also suggests that sheep, in which it is more pathogenic than F. hepatica sugar cane grown in swampy areas may serve as a (OGU RINADE, 1979), inducing a higher mortality and 186 S. MAS-COMA & M.D. BARGUES hence a greater economic problem (OGU RINADE, ding mice, rats, rabbits, sheep, goes and cattle. These at- 1984a; AlA USSI et aI., 1988). The greater pathogenicity tempts comprised sensitization by primary homologous has been related to the longer migration period through or heterologous normal or irradiated infections per os, the liver parenchyma (EL HARITH, 1980; OGU RI ADE, sensitization by subcutaneous, intramuscular or intrape- 1984b). ritoneal implantation with the various fluke stages, sen- Similarly as with F. hepatica, the association of F. st- sitization by somatic extracts or metabolic products of gantica with Clostridium infection (Clostridium novyi mature or immature flukes and passive transfer of resis- type B) also leads to infectious necrotic hepatitis (Black tance by immune serum or sensitized Iymphocytes. disease) among African sheep (ABU-SAMRA et al., 1984). Prevention and control Diagnosis Owing to the similarity of the life cycles, prevention The etiologic diagnostic methods and aspects to be ta- and control measures follow the same patterns as for F. ken into account are similar to F. hepatica. The possibi- hepatica. The peculiarities of F. gigantica must, howe- lity of spurious fascioliasis due to passing of transient ver, be taken into consideration. Thus, in enzootic areas eggs after eating infected cattle or sheep liver must be of F. gigantica, contraction of the infection by the ani- verified. In the not uncommon case of failure to find mals and their contamination of the area with eggs of the eggs in faeces, egg search can be made in liver aspirate parasite in the faeces take place when the animals go to (PERRY, GOLDSMID & GELFAND, 1972). drink, rather than when they are grazing in the pasture. Among immunological tests, immunoelectrophoresis Accordingly, avoiding the watering of the animals from was used by MOREAU et al. (1975), counterirnmunoelec- swampy banks of rivers and from bodies of water rich in trophoresis by MA SOUR et al. (1983), and passive hae- vegetation would considerably reduce the chances of in- magglutination and electrosyneresis (counterimmunoe- fection. lectrophoresis) by MAIGA et al. (1991). HILL YER (1988) A molluscicide such as N-trytylmorphine at a concen- emphasized ELlSA as an extremely sensitive method, tration of 0,09-0,1 parts per million allowed the eradica- becoming seropositive by 2 weeks postinfection and se- tion of the snail host for II months (PRESTO & CASTE- ronegative after therapy, in which cross-reactivity of LINO, 1977). crude extracts with other parasitic infections have been Worth mentioning are the studies by GUPTA, PRASAD resolved by gel filtration in the case of F. gigantica & CHA DRA (1986) on the possibilities of biological (MANSOUR et al., 1983). YOUSSEF & MA SOUR (1991) control of the snail host of F. gigantica. These authors evaluated partially purified F. gigantica antigens in an observed that Channa punctatus, a freshwater fish which ELlSA for the specific serological diagnosis of fasciolia- may be cultured in derelict, weed-infected waters, feed sis. The diagnostic potential of 10 antigen fractions deri- on L. auricularia. Laboratory studies showed the rates of ved from gel-filtration peaks (II and Ill) were evaluated predation to vary with the size of the fish and of the mol- in patients with Fasciola, Schistosoma, Entamoeba and luscs. Echinococcus. Fractions 2 and 10 were highly specific for Fasciola infection and failed to react with other para- sites. It is considered that the ELlSA should be conside- DICROCOELIUM DENDRITlCUM red as a rapid and specific technique for the diagnosis of fascioliasis and also as an epidemiological surveillance Morphology technique in areas endemic for F. gigantica infection. This species is popularly known as the lancet fluke, the lanceolate fluke, the little liver fluke or the small liver Treatment fluke. The adult stage has an elongate body, which is na- In animals, the same compounds as for F. hepatica are rrow anteriorly, and the widest portion is behind the used (rafoxanide, oxiclozanide, brotianide, niclopholan, middle, 8-1412-2,5 mm in size.Host-related size differen- niclosamide, hexachlorophene, etc.). The same can be ces have been observed (FETISOV, 1978). The tegument said for man. In man, treatment with metronidazol and is mooth, without spines. The oral sucker is smaller than emetine hydrochloride was apparently successful the acetabulum, which lies in the first third of the body (PERRY, GOLDSMID & GELFA D, 1972). Recently, length. The pharynx is small and the caeca do not reach SUHARDONO et al. (1991) applied triclabendazole in In- the posterior body end. The testes are postacetabular, jux- donesian cattle for the control of F. gigantica at a dose of tapose or tandem in position, sometimes asymmetrical, 12 mg/kg orally every 8 weeks for I year; egg count in frequently lobed. The ovary is postesticular. The two la- treated animals was significantly reduced to almost zero. teral fields of minute vitelline follicles are situated extra- Concerning a future possible vaccine, HAROUN & caecally in the mid-body. The uterus starts at the ootype HILLYER(1986) reviewed attempts to actively stimulate about the middle of the body, and its ramified transverse or passively transfer resistance to Fasciola, including F. coils occupy the central field from the middle of the body gigantica, in various laboratory and farm animals, inclu- to the posterior end, ascending and descending uterine Human liver flukes: a review 187 branches traversing one another. An ascending branch & LEGER, 1967; MA DOUL et al., 1966; VERMEIL et al., proceeds to the postbifurcal, median genital pore, in 1964), Italy (V A UCCHI & RICCARDI, 1962), Sweden which a prominent cirrus pouch also opens. The embryo- (BE GTSSON et al., 1968), Germany (SCHEID, MEND- nated eggs are dark brown, thick-shelled, operculated at HElM & AMENDA, 1950), Switzerland (GALLI- VALERIO one pole, and measure 35-45/22-30 um. & BORNAND, 1931; STAHEL, 1981), Spain (VASALLO MATILLA, 1971a, b), Hungary (LORINZ, 1933), and Ru- mania (SCHEID, MENDHEIM & AMENDA, 1950); and D) Location and definitive hosts South America:Brazil (TRAVASSOS, TEIXEIRA DE FREI- The adult of this species is a parasite of the bile ducts TAS & KOHN, 1969). In the past, several post-mortem and gall bladder. It shows a scarce host specificity, accor- examinations disclosed the presence of D. dendriticum in ding to the more than 60 mammal species of the orders the liver of man in Germany, Rumania, Armenia, Egypt Rodentia, Lagomorpha, Artiodactyla, Perissodactyla, and China (SCHEID, ME DHEIM & AMENDA, 1950). Carnivora and Primates in which it has been reported so Reports on true human infections published in recent far. Additional new host records have appeared in recent years concern different countries, such as Czechoslova- years (i.e. GVOZDEV & ORLOV, 1985). However, only kia, Turkey, Uzbekhistan (old USSR), Iran, Saudi Ara- members of the suborder Ruminantia, notably sheep and bia, and the USA. In Czechoslovakia dicrocoeliasis in cattle, may be considered as true definitive hosts, remai- man is reported for the first time by LOFAYOVA, CATAR ning definitive host species, including humans, being & HOLKOVA (1987). In Turkey, KILI<;:TURGAY et al mere alternate hosts according to the relatively low inci- (1982) reported D. dendriticurn at 0,96% among 6311 dence and intensity of infection in them. patients, and COSKU ER et al. (1979) found seven ma- Numerous species have been used as definitive hosts ture D. dendriticum in a 26-year-old woman after a gall for experimental purposes: the Syrian hamster proves to bladder operation. In Uzbekhistan, old USSR, D. dendri- be the better host, and the golden hamster and rabbit are tiCU111 adults were found in the livers (up to 4 flukes/li- good hosts; other species used are cotton rats, white rats. ver) of 37 of the 13287 corpses autopsied from 1968 to guinea pigs, rabbits, sheep, cows, dogs, cats, monkeys, 1986; in no case was death caused by the infection, nor and white mice (HOHORST & LAMMLER, 1962). Experi- was the infection ever diagnosed intra vitam (AZIZOVA mental infections proved the wide range of hosts which er al., 1988). In Iran, two cases were reported by SOH- become infected with this fluke (SALlMOV, 1973). RABI (1982/1983). In Saudi Arabia, D. dendriticum was found in the sto- ols of seven Saudis, of which six patients complained of Reports in humans abdominal pain and other gastrointestinal problems; D. Human infections have been recorded in D. dendriti- dendriticum was consistently found in the faeces of a 30- cum enzootic areas. Although the majority of these infec- year-old male patient with hepatomegaly (BOLBOL, tions are of rare and sporadic occurrence, they are un- 1985). Also in Saudi Arabia, 208 patients were found doubtedly underestimated. Most infections are only excreting D. dendriticum eggs. in 1984-1986.Peak inci- spurious (STAHEL, 1981; BERNHARD, 1985; KHA et al., dence occurred between October and ovember in each 1988; MOHAMED & MUMMERY, 1990; etc.), the appea- year. At least 7 had true infections, 134 patients were rance of eggs in stools being due to the ingestion of infec- symptomatic (mainly eosinophilia, right hypochondrial ted livers of sheep, goat and cattle, eggs being detected in pain, flatulence and diarrhoea), 16 patients had disturbed faeces for only a few days. The reasons for the availabi- liver function, 13 had gall bladder or biliary tree disease, lity of infected animal livers on the market is that not all and this was clinically considered to be caused by D. infected livers show signs of the infection. Genuine hu- dendriticum in at least 2 patients (MOHAMED & MUM- man infection can be verified through parasite finding in MERV, 1990). In the same country, findings of eggs in fe- surgical operations, evidence of permanent egg shedding aces have been also recently described by KHAN et al. through time, egg recovering in duodenal aspirates, or the (1988) and OMAR, ABU-ZEID & MAHFO Z (1991). existence of related symptomatology. In the USA, DRABIK et al. (1988) reported a case of D. Human cases were reported long ago from: A) Asia: dendriticum infection considered to be genuine in a 23- USSR (ASSATOUROW, 1931; GIGITASHVILI, 1962; JAGU- year-old homosexual man from northern New Jersey, BOFP, 1929; KALANTARIAN, 1926; MTSCHEDLlDZE, USA, who was found to be HIV seropositive in Septem- 1931; PIGOULEWSKY, 1927a, b; V ASILlEVA, 1927), Iran ber 1986. Faecal examination revealed 5 ova of D. den- (FARID, 1971), Java (mentioned in ROSICKY & GROS- driticum. Before the possibility of a spurious infection, CHAFf, 1982), and China (SCHEID, MENDHEIM & false infection was excluded only through information AME DA, 1950); B) northern Africa: Lebanon (COMBES- provided by the patient about his diet. Authors were una- COT, MAMO & AKATCHARIAN, 1973 in TOHME & ble to obtain further stool specimens since the ova were TOHME, 1977), Syria (YENIKOMSHIAN & BERBERIA, only directed after the patient had been discharged from 1934), Egypt (SCHEID, MEDHEIM & AME DA, 1950). hospital; authors stress that the patient had vehemently Tunisia (BOURGEO et al., 1974); C) Europe: France (SI- denied ever consuming liver. They considered that the GUIER et al., 1952; LAVIER & DESCHIE S, 1956; CA VIER small number of ova observed is more consistent with 188 S. MAS-COMA & M.D. BARGUES true infection and conclude that it is a genuine case. The maged after complete digestive crossing, so that when infection is thought to have been acquired through gi ven to appropriate snailsthey continued development. found floating in bottled drinking water supplied at the Miracidia hatched inside the snail intestine migrate patient's work place in ew Jersey. through the gut wall to the adjacent connective tissues where they metamorphose to mother sporocysts. These migrate to the digestive gland or hepatopancreas where Geographical distribution they develop. Each mother sporocyst gives rise to seve- This parasite species has a more or less cosmopolitan ral cercariogenous daughter sporocysts. Mature daugh- distribution in herbivorous mammals, mainly in rumi- ter sporocysts, sac-shaped, 1,2-3,5 mm in length (MAT- nants of the Holarctic region.Thus, sheep and cattle-rai- TES, 1936; KRULL& MAPES, 1952c), can be numerous sing countries are menaced by dicrocoeliasis due to D. in a host individual depending of its size: 396 in a 4,5 dendriticum. This species is common in almost every mm long snail, 587 in a 6,5 mm snail (MATTES, 1936). country and adjacent islands of the European continent The time necessary for sporocyst development differs and is also found along the northern coast of Africa. It according to the molluscan species and environmental has been said to occur in South Africa, but such reports conditions. Sporocysts are first observed from 50 days have not been corroborated. In Asia it is found in the old postinfection (d.p.i.) in Zebrina detrita (N6LLER, 1932), USSR, especially in Siberia and Turkestan, as well as in 70-80 d.p.i. in Cionella (= ) lubrica (MAPES, Turkey, Syria, Iran, India, China, the Philippines, and 1951; BADIE&RONDELAUD,1990), or 123 d.p.i. in He- Japan. In the Americas, the fluke is found in the USA licella (Helicopsis) arenosa (TIMON-DAVID, 1965). and Canada in North America and in Cuba, Colombia Stylet-bearing, long-tailed, xiphidiocercous cercariae, and Brazil in Central and South America. 360-760/51-164 urn in size (tail 0,20-1,0 mm long), origi- In the USA, animal dicrocoeliasis was detected during nally described as Cercaria vitrina by YOGEL(1929), are meat inspection at Newark, New Jersey, in cattle coming produced inside these second generation sporocysts and from New York state (PRICE& KI CHELOW, 1941). exit through a birth pore. The production of the cercariae Subsequent reports showed that the infection occurred in is slow, and it requires about 3 months. The number of cattle and sheep in several countries of the state of New cercariae simutaneously present in one sporocyst ranges York and that the infections could sometimes be exten- from 10 to 40 (MATTES, 1936; NEUHAUS, 1936). They sive. In the same enzootic area, infections were reported are expelled by the snail in some mucus when there is a from goats, horses, the white-tailed deer Odocoi/eus vir- drop in the temperature. A mass of these cercariae in mu- ginianus, the wood-chuck Marmota mona, and rabbits. cus is known as a «slime ball» (matrix surrounded by a The parasite is believed to have been introduced with the layer of clear, gelatinous material, all forming a spherical extensive importations of dairy cattle into New York structure). The number of cercariae contained (280-460) state from European countries. and size of the slime balls (1-3 mm in diameter) differ ac- In Canada, the first report of D. dendriticum was in cording to the snail hosts (NEUHAUS, 1938; KRULL & 1930, in sheep from Nova Scotia and Quebec. Subse- MAPES, 1952a; GROSCHAFT,1959). Slime balls offer cer- quent reports were again from eastern Canada until 1974 cariae an appropriate aquatic environment for the transit when it was found in cattle in British Columbia, western between the terrestrial snail host and the second insect in- Canada (LEWIS, 1974). termediate host. The slime balls are released from the snails individually or in clusters of 4 to 16. A production of up to 5 slime balls per snail in 23 days was observed Life cycle by KRULL& MAPES(1952a). The life cycle of D. dendriticum follows a triheteroxe- Ants, foraging over leaves and sticks where the snails nous pattern. Embryonated eggs expelled by adult live, find the slime balls and carry them to their nests. worms pass through bile ducts and intestine and are ex- Whether the adult ants eat the slime balls or feed them to creted with the faeces, to follow a completely terrestrial larval ants is not known. Inside the ants, the cercariae life cycle. Hatching of the eggs deposited on the soil migrate through the gut wall and encyst in the abdominal does not occur until ingested by appropriate land snails, cavity and infrequently the other parts of the body of the which act as the first intermediate host. A total of20% of to form encysted metacercariae (SCHNEIDER& Ho- hatched eggs are found in the snail's intestine 20 minu- HORST,1971). Fully formed metacercarial cysts are oval tes after initial infection (RATCLIFFE,1968). There appe- (228-440/192-328 urn), relatively firm, thick-walled (10- ars to be no relationship between egg dose used and per- 40 urn), resilient, and only slightly sticky, resembling centage of eggs hatching in molluscs (ALU DA & thick gelatin (KRULL&MAPES, 1953a). The develop- ROJO-YAZQUEZ,1982), and some molluscs which elimi- ment within the ant up to the ripe metacercarial stage re- nate hatched eggs with the faeces are afterwards not quires at least 38-56 days at 26° C (YOGEL & FALCAo, found to be infected (MAPES, 1951; TARRY, 1969; KAL- 1954). Reports on metacercaria number per ant varies KAN, 1971). SIDDlKOV(1986) has demonstrated the role considerably. As many as 580 cysts have been found, of beetles and grasshoppers as disseminators of D. den- but the average is usually lower than that: 1-5,1-120,6- driticum eggs, a small proportion of which remain unda- 80,17-74, up to 370, 7-107, 2-304, etc. (KRULL& MA- Human liver flukes: a review 189

PES, 1953b; KLESOV & POPOVA, 1956; GROSCHAFf, TOHME(1977) and Cochlicella ventricosa (BADIE & 1961; HOHORST, 1962; etc.). Ro DELAUD, 1987). It is evident that D. dendriticum is When infected ants are swallowed with the food by the able to adapt to different species depending on mammalian host, the metacercariae ex cyst in the duode- the geographical area, a capacity which is not usually num (the protective envelope is softened by the digestive found among Digenea in general and which has allowed juices, mainly trypsin and bile, of the host). As verified this parasite to spread up to its actual very wide geograp- in experimental infections, the parasite (540-635 urn hical distribution. It must be taken into account, howe- long) enters the common bile duct and reaches the bi- ver, that reports of several species considered to be the liary tree in about an hour after ingestion (KRULL&MA- snail hosts of D. dendriticum were based only on cir- PES, 1952b; KRULL, 1958). Although rejected by several cumstantial evidence, because they were the only spe- authors (KRULL, 1958; SOGOY A , 1960; LAMMLER, cies encountered in the area surveyed. 1962), the other suggested alternative way, entering the intestine wall and being carried passively to the liver by Second intermediate hosts means of the portal blood system, cannot be excluded, so that both modes may exist simultaneously (ALlEV, 1966; The various ant hosts have been tabulated in a recent ROSICKY & GROSCHAFf, 1982). Sexual maturity is report, together with their geographical distribution and achieved in about 6-8 weeks (TARRY, 1969; CHANDRA, the number of metacercarial cysts encountered (SRIVAS- 1972; FETISOV, 1978). The prepatent period slightly dif- TAVA, 1975; ROSICKY & GROSCHAFf, 1982). Of the 17 fers according to the host species: 47 days in lambs, 44- species of ants reported infected with the rnetacercariae 50 days in rabbits, 50 days in calves, 52 days in kids, and of D. dendriticum, 14 species are members of the genus 54 days in donkeys. The infection capacity of metacerca- Formica, including Ffusca, F cinerea, F clam, F cuni- riae pronouncedly differ according to host species: cularia, F gagates, F lugubris, F meroasiatica, F ni- 32,4% in donkeys, 50% in kids, 60,6% in lambs, 56,7% gricans, F picea, F polyctena, F pratensis, F rufibar- in calves, and 21,3% in rabbits. bis, F. sanguinea and F subpilosa. The remaing three D. dendriticum may survive in the definitive host for intermediate host species are Proformica nasuta, Cata- greatly prolonged periods. According to Ko 0 ov gliphis bicolor and C. aenescens. (1963), it may survive for as long as 8 years in a ram Interesting behavioural changes are produced in ants kept in isolation. as a result of their infection with D. dendriticum meta- cercariae. Some cercariae invariably become encysted in the brain of the ant and cause abnormal behaviour First intermediate hosts which enhances the chances of ingestion of the ant by According to ROSICKY& GROSCHAFf (1982), up to 54 the grazing mammalian host. The metacercariae in the land snail species have been identified so far to serve as brain, mainly in the suboesophageal ganglion, causes first intermediate host: : Cochlicopa lu- paralysis of the mandibles, and thus, the ant remains fi- brica; Pupillidae: Abida frumentum; : Chondrula xed to pieces of vegetation at various times of the day tridens, Jaminia potaniniana (= Subzebrinus p.), Ena (HOHORST& GRAEFE, 1961; HOHORST, 1962; BADIEet obscura, Jaminia sieversi, Zebrina hohenackeri, Z. de- al., 1973). Apparently, there is a diurnal rhythm exhibi- trita, Subzebrinus sogdianus, Pseudonapaeus miser, Ch. ted by the paralyzed ant (called periodic catalepsis): the microtraga ("), Pseudochondrula brevior, P. schelkovni- number of cataleptic ants is high in the morning and kovi; Claisilidae: Armenica brunae, Laciniaria varnensis; evening and low during midday. Temperature, and not Zonitidae: Zonitoides nitidus, Oxychilus derbentinus; light, seems to be the environmental factor regulating Ariophantidae: Macrochlamys cassida ("), M. monticola; this rhythm. The daily rhythm followed by the catalep- Eulotidae: Eulota lanizi, E. macci,E. duplocincta, E. tic state of the ants seems to coincide with daily activi- phaeozona,E. fruticum, E. paricincta, E. almaatini, E. ties of grazing animals. The latter are active in the mor- rubens, E. semenovi, E. skwortzowi, E. plectotropis t= ning and evening but are inactive and seek shady areas Cathaica plectotropis); Helicidae: He/icella candicans, at midday (ANOKHI , 1966; SPINDLER, ZAHLER & H. candaharica, H. itala, H. candidula, H. crenimargo, Loos-FRA K, 1986). H. derbentina, H. unifasciata, H. virgata, H. krynickii, H. protea (?), H. schelkonickovi, Theba carthusiana, Zeno- Epidemiology biella rubiginosa, Fruticocampylaea narzanensis, Tri- chia eichwaodi, E. selecta, Metafruticicola pratensis, Ce- In some enzootic areas, infection rates with D. dendri- paea nemoralis, Helix vulgaris, H. lucorum, Trochoidea ticum in the snail hosts may be high.From 10% to 20% pyramidata, Cochlicella acuta. of the snails H. candidula and Zebrina detrita were infec- In another review made by ALU DA (1984), several ted in Germany (VOGEL, 1929). More than 24% of Cio- other species are added. And there are even snail species nella lubrica were infected in ew York, infection rates not mentioned in the list of ROSICKY& GROSCHAFf varying according to the month (17, 18,24, and 18% for (1982), nor in that of ALUNDA(1984), such as Theba sy- the months of June, July, August, and September, respec- riaca and Xerophila vestalis detected by TOHME & tively) (KRULL &MAPES, 1952c). In sheep pastures in 190 S. MAs-CoMA & M.D. BARGUES

Germany, it was found that the majority of specimens of Examples of prevalence rates of the infection of cattle Helicella obvia, become infected in the autumn of their in different countries are: 46-65% in Switzerland (Ec- second year of life, when their shell diameter was of me- KERT, SAUERLANDER& WOLFF, 1975), 0,7-100% in dium size. The percentage of snails containing daughter Spain, 5-60,6% in Italy, 0,8-26% in Sweden, 0,5-21 % in sporocysts was highest in spring. Slimeball output could Bulgaria, 2,7% in Russia, 30% in Azerbaidzhan, 1,51% be provoked only in May and June (SCHUSTER, 1993). in Pakistan, and 27-35% in Lybia (see review in GONZA- The formation of the slime balls is associated with a drop LEZ-LANZA, MANGA-GONZALEZ & DEL-POZO-CAR- in the temperature in the snail environment (KRULL & NERO, 1993). In sheep, up to 100% has been recorded in MAPES, 1952a; VOGEL& FALCAO,1954). Bosnia and Herzegovina, with fluke burdens averaging High prevalence rates of infection among ants are also 1650-2837 (even more than 14000 worms) per animal on record: 31% in New York (KRULL& MAPES, 1953b); (RUKAVINA,1977). In cattle, infection prevalence usually 33% in Bulgaria (DENEV et al., 1970); 83,4% also in increases with age. Monthly average numbers of eggs per Bulgaria (SRIVASTAVA, 1975). Moreover, rates vary gram of faeces in Spanish cattle ranged from 26 to LOO in both seasonally and from year to year: in France 100% cattle.Seasonal differences in egg elimination appear ac- and 35% in June and July 1973, respectively, and 100%, cording to different regions (GONZALEZ-LANZA,MAN- 13% and 30% in April,August and September 1974, res- GA-GONZALEZ& DEL-POZO-CARNERO,1993). pectively (BADIE, 1976). There are considerable varia- That dicrocoeliasis occurs in a number of reservoir tions in the incidence of metacercarial infection in the hosts is an important aspect in the epizootiology of the ant intermediate host depending on the site where ants disease. In addition to sheep and cattle, it has been natu- are collected and the labour organization in the ant co- rally found in goats, deer, elk, rabbits, and pigs.Rabbits lony. The incidence of infection was reported to be only seem to intensify the infection in local areas (BAILEN- 2% among ants that were collected directly in the nest, GERet al., 1965), and deer in their wider movements dis- while 43,3% of the ants that were collected outside the seminate it. Moreover, the practice of shipping heep nest, in the pasture, were infected (SVADZHYAN,1956), and cattle from one area to the other for new pasture also SPINDLER,ZAHLER& Loos-FRANK (1986) studied the helps in the spread of the infection, especially because of behaviour of naturally-infected Formica spp. on a steep. the wide distribution of the snail and ant intermediate On a given day, 36% of the infected ants in the meadow hosts (ROSICKY& GROSCHAFT,1982). were attached to grasses, 9% to bushes and 55% to flo- Humans acquire the infection accidentally (by swallo- wering plants. On the slope the corresponding percenta- wing an infected ant together with the food, such as ve- ges were 9, 13 and 77. A total of 74% preferred the blos- getables, fruit, etc.) while staying in the endemic area. som of flowering plants, 24% the leaves and 2% Thus, the principal human source of dicrocoeliosis beco- preferred the stalks. The lowest number of metacercariae mes infested sheep with extremely high worm loads (up found in the ants was one; 32% contained less than 20 to 108000 worms), and a very high level of incidence of metacercariae. The study showed that most attached ants infection (often 100%). The consequence is a high rate leave the plants during the day, that ants do not readily of pasture resulting from faecal contamina- die from starvation, and that in conditions of high tempe- tion, which is in turn potentiated by large-scale, inten- ratures and low humidity ants die regardless of whether sive breeding of these animals. The great quantity of or not they are infected. eggs laid by a single fluke during its several-year life- Infections in ants are apparently of long duration, ants span is multiplied many times by the larval stage multi- probably harbouring mature and viable cysts throughout plication at snail host level. This is responsible for the their life span, although the infection is believed to shor- heavy infestation of the pasture environment, especially ten the life span of the ant. It has also been noted that the under favourable conditions. Another important epide- metacercariae evidently overwinter in the ant (BADIE, miological feature is the fact that the cercariae are inter- 1976). The overwintering of the infection in the ant, as mittently shed by the snail intermediate host at short in- well as the diurnal rhythm of the cataleptic state of the tervals dictated by sudden cJimatological changes, such infected ants, are factors of significance in the epizootio- as decrease in atmospheric pressure and temperature, logy of the disease, because they enhance the chances of and increase in relative humidity notably during thun- the mammalian hosts becoming infected. derstorms (ROSICKY& GROSCHAFT,1982). Despite the very limited capacity of metacercariae for successful mammal infection (approximately 30% of in- Pathology, symptomatology gested rnetacercariae continue their development in the and clinical manifestations definitive host, after VOGEL & FALCAO, 1954), adult burdens are usually large because of the cumulative na- D. dendriticum infection is an important parasitic dise- ture of infections, which increases the number of eggs ase in animals from an economic and health viewpoint being discharged in the faeces and thus ensures the (LUKIN, 1980; WOLFF,HAUSER& WILD, 1984). As with maintenance of the life cycle. Infected sheep in some other liver fluke infections, the pathology depends on the areas might harbour up to 7000 worms in the gall blad- number of flukes present and the duration of the infec- der and 50000 in the liver. tion. Because of the small size of the fluke and its smo- Human liver flukes: a review 191 oth and spineless surface, mechanical and toxic damage of 48%, which was maintained at levels of 58 and 50% are much less than in fascioliasis and opisthorchiasis. (SIGUIERet al., 1952). Also worth mentioning here is that the main route of mi- It is assumed that the same pathological changes occur gration of D. dendriticum via the bile ducts differs from in humans as in animals. The extent of liver changes that of F. hepatica, which reaches the liver via the abdo- conform to that described for infected animals (see a de- minal cavity route. tailed description in ROSICKY & GROSCHAFf, 1982). The incidence of infection and intensity of the clinical Worth mentioning in animals is that a relationship with manifestations are dependent on the frequency of expo- bile duct carcinoma has never been reported. sure and the length of time that the population has been exposed to the infected environment. These are likewise Diagnosis dependent on the degree of infestation in the environ- ment and the prevailing conditions and practice of hy- Coprological methods, for egg finding in faeces, pro- giene by the population living in the vicinity of potential vide sufficient diagnostic evidence and are technically dicrocoeliasis foci. undemanding. Satisfactory diagnostic results have been Approximately 300 cases of human dicrocoeliosis, reported with the use of modified flotation and sedimen- identified at least on the basis of finding of eggs in the tation methods. In heavy infections, examination of a di- stool, were reported in the literature up to 1982 (Ro rect faecal smear is adequate to demonstrate the presence SICKY & GROSCHAFT, 1982). However, this number of characteristic eggs. The «cellophane faecal thick- may not be accurate since: A) dicrocoeliasis may be smear technique» or simply «Kato-Katz method» accor- confused with infections of different etiology;B) for the ding to KATO & MIURA(1954) modified by KATZ,CHA- most part of reported cases, absence of false infection is VES& PELLEGRINO(1972) is recommended because it is not demonstrated (eggs merely passed through the intes- rapid, inexpensive, reproducible and allows quantitative tinal tract following the consumption of infected livers); as well as qualitative detection of eggs. C) true dicrocoeliasis usually appears to be symptom- The finding of D. dendriticum eggs in the faeces of hu- less, so that cases are simply not diagnosed because af- mans and carnivores does not necessarily indicate a true fected persons do not go to the specialist (when appa- infection with this parasite. The presence of eggs in the rent, clinical symptoms may suggest a hepatitis-like faeces of these hosts could follow the consumption of in- infection). fected livers resulting in spurious infection. To establish In man, clinical symptoms of true dicrocoeliosis are diagnosis of a true infection, the stool should be exami- neither uniform nor specific. Generally, the infection is ned at intervals and shown to be repeatedly positive. It accompanied by either a prolonged period of constipa- should also be complemented with a serological test or tion or diarrhea, nausea, and vomiting. Some patients an adequate clinical examination. In some instances, true may complain of abdominal discomfort and pain in the dicrocoeliosis can also be distinguished from spurious right half of the abdomen and in the epigastrium radia- infection in that, in the former, the eggs in the faeces are ting to the right shoulder. Sometimes, the disease is ac- all embryonated and of a dark brown colour (ROSICKY& companied by lassitude, headache, and giddiness. Pain GROSCHAFf, 1982). For the recovering of eggs, duode- in the liver is continuous, independent of the uptake of nal aspirates have also been used in genuine human ca- food, and intensifies mainly at night. Objective signs in- ses (ASSATOUROW,1931). clude subicteric colouration of the cornea and the skin Sero-diagnostic methods, such as complement-fixa- and enlargement of the liver and the spleen. In the initial tion-test, precipitation test, and skin tests, have also been stages, there is leukocytosis, eosinophilia (8-25%), and used more or less successfully. occasionally, traces of bile acids in the urine. Later on, slight anemia may ensue; the leukocytosis drops to nor- Treatment mal level and eosinophilia diminishes to 5 to 7% (PRICE & CHILD, 1971; ROSICKY& GROSCHAFf, 1982). Human dicrocoeliasis has received little attention on Cerebral involvement in dicrocoeliasis is rare, but a account of the small (a few hundred) number of cases so French shepherd aged 17 years, infected with D. dendri- far encountered. Thymol was one of the first drugs to be ticum, showed a brain condition apparently due to dicro- used against dicrocoeliasis in a few human cases. Hetol, coeliasis. The body developed convulsive crises and Hetolin® (Hetol-D), thiabendazole, and their analogs are Jacksonian gait, affecting the left-hand side, with slight the commonly used anthelminthics for dicrocoeliosis symptoms of deficiency in that side. Several days later which cause no side effects on the host. Dicrocoeliosis in he had a hemiplegia on the right-hand side, and finally a humans, as well as in animals, has been more or less suc- meningeal syndrome was imposed on these symptoms. cessfully treated with these drugs (GUILHON, 1956; Evidently, D. dendriticum or its eggs had been carried to LAMMLER, 1964a, b, c). Human dicrocoeliosis has hit- the brain via the blood and caused these neurological herto been treated mainly with emetin and Entobex (CA- symptoms. In addition to eggs of the parasite constantly VIER& LEGER, 1967; VERMEILet al., 1964; CAVIER& in the boy's faeces, he was positive for an intradermal ERHARDT, 1973). More recently, BUNNAG&HARINA- test using «distome» antigen. There was an eosinophilia SUTA(1989) recommended praziquantel, in doses of 25 192 S. MAS-COMA & M.D. BARGUES mg/kg 3 times daily after meals for 1 or 2 days. This The administration of suitable anthelminthics to infected drug has already been used in human dicrocoeliasis animals has an adjuntive value in the prevention and con- (DRABTKet al., 1988; MOHAMED& MUMMERY, 1990), trol of dicrocoeliosis and enhances the efficacy of the pre- as well as in animals (GURALP,OGUZ& ZEYBEK, 1977). ventive measures applied. A satisfactory measure to pre- If praziquantel is not available, bithionol, 15 to 25 mg/kg vent debilitation or death of animals infected in the spring twice daily on alternate days for 10-15 days may be ef- and to limit the degree of infestation of the pastures is the fective (MARKELL& GOLDSMITH,1984). administration of anthelminthic drugs in between pasture Thymol, Fouadin®, thiabendazole, hexachloroparaxy- seasons (FETISOVet al., 1970; TvERDOCHLOBOV,1971). 101,hetol (effective against the sexually mature flukes), Hetolin® (effective against the young, sexually imma- ture flukes), and diamphenetide are drugs having been DICROCOELIUM HOSPES successfully used against animal dicrocoeliasis (RO- SICKY& GROSCHAFf, 1982). Combinations of Hetol or Morphology hexachloroparaxylol and Hetolin® (FETISOV, 1971a, b) or thiabendazole and Hetolin® (DELIC, CANKOVIC& The adult stage of D. hospes is similar to that of D. den- ROZMAN,1971) proved to be even more effective than driticum, but differs in presenting a more slender, slightly the same drugs used separately. smaller (4,5-12/0,5-1,4 mm in length/width, and thus a different ratio of body length to width) body of an almost uniform width except for the tapering anterior portion, Prevention and control ventral sucker lying somewhat closer to the anterior end The control of dicrocoeliasis is similar to that of several of body, lobed, tandem or slightly oblique testes, vitellaria fluke infections, although taking into account that the in- in larger follicles occupying a compact and smaller area, termediate hosts are terrestrial snails and ants. Preventive being intracaecal and always posterior to the ovary, uterus measures performed to control the intermediate host po- less ramified with descending uterine branch on the ova- pulations in the pasture should be extended to the entire rian side of the body and ascending branch on opposite endemic area, because otherwise foci of dicrocoeliosis side, the two branches not crossing each other. The eggs would persist. Altering the character of the infested pastu- are embryonated, dark brown, with thick walls, opercula- res must be the first step to reduce the intermediate host ted at one pole, and measure 35-45/20-30 um (GRABER& populations. Periodic changes of the planted grasses lead OUMATTE,1964; BOURGAT,SEGUIN & BAYSSADE-Du- to unfavourable conditions for the survival and multiplica- FOUR,1975; KAJUBIRI& HOHORST,1977). tion of the intermediate hosts and simultaneously improve An interesting study on variability range and fre- the quality and productivity of the pasture (KLESOV& Po- quency of occurrence of morphological types, carried POVA,1956; GROSCHAFT,1959; HASSLER,1963). out by MACKO& PACENOVSKY(1987) by comparing D. Land snails can be controlled biologically, physically, dendriticum materials from Algerian and Czechoslovak or chemically. For the biological control, chickens have cattle, lead the authors to think that D. hospes could be been successfully used in areas of the old USSR, as they interpreted as an intrapopulation morph of D. dendriti- feed voraciously on land snails, thus considerably redu- cum and its junior synonym. cing their populations in a short time (PETROCHENKO& TVERDOKHLEBOV,1971). Certain physical methods, Location and definitive hosts such as hand picking and crushing, have been employed against terrestrial snails with great success in some pla- This species is an African parasite of the biliary ducts ces. Chemical measures, such as the use of repellents, at- and gall bladder. Recorded definitive hosts are sheep, tractants, or contact poisons, have also been applied with cattle, zebus, goats, and monkeys from the Ethiopian re- some success. Of course, care must be taken in the use gion (ROSTCKY& GROSCHAFT,1982) and handling of certain types of chemicals because they may be poisonous to man and animals and may be inju- Reports in humans rious to certain plants on the pasture. Concerning ants, effective chemical preparations are D. hospes has several times been reported as a human available for chemical control. Satisfactory results have parasite. Several reports of spurious human infection been obtained using a combined biological and chemical have appeared, such as in Ghana (ODEI, 1966; WOLFE, methods. An aqueous emulsion 0,35% of dicresyl ester 1966) and Kenya (CHUNGE& DESAI, 1989). Such hu- at a dose of 200 to 250 ml/nr' was sprayed on sites colo- man infections appear to have resulted from consump- nized by the ants. The chemical paralyzes the ants and tion of infected sheep, goat and cattle livers containing they are in turn picked up by the flocks of free chickens adult worms, with subsequent appearance of eggs in hu- (SALTMOV1970), . However, in some areas, the use of in- man stools but only for a few days. The reason for the secticides to kill the ant hosts is objectionable or even availability of infected animal livers on the market is forbidden, because of the possible contamination of the that not all infected livers show signs of the infection; environment. and in Ghana, for example, only about one third of these Human liver flukes: a review 193 are condemned due to the presence of gross pathology Life cycle (WOLFE, 1966). In Senegal and Mali, MALEK (1980) en- countered D. hospes in livers which had already been The life cycle of D. hospes is similar to that of D. den- inspected and were on their way to the market. driticum. The miracidium of D. hospes is ovoid, measu- Genuine human infections have been reported in Zaire ring 30-35/23 urn, Terrestrial snails act as first interme- (former Belgian Congo) (VAN DENBERGHE & DENECKE, diate host in which cercariogenous sporocysts develop. 1938), Ghana (ODEI, 1966), Sierra Leone (KING, 1971) The body of the cercaria of D. hospes measures 470- and Nigeria (ROCHE, 1948; HARMON & OYERINDE, 1976; 550/150-170 urn. The tail measures 600-700/150-170 urn REINTHALER et aI., ] 988).V AN DEN BERGHE & DE ECKE in length/maximum width. The argentophilic papillae of (1938) reported one case and ODEI (1966) two cases. the cercaria of D. hospes are very similar in number and KI G(1971) reported D. hospes infections in 15 persons, location to those of D. dendriticum. Metacercariae deve- including 10 Americans and 5 who had never left the lop in ants (BOURGAT, SEGUI & BAYSSADE-DuFOUR, country; all were residents in cattle-raising areas and two 1975; LUCIUS, ROMIG & FRA K, 1980). Metacercariae of them appeared to be heavily infected. ROCHE(1948) re- are ovoid, 174/120 urn in size, and are found at a rate of ported one human case due to D. hospes (identified in the up to 363 metacercariae per ant (ROMIG, LUCIUS & report as D. dendriticums. HARMON & OYERINDE(1976) FRANK, 1980). found eggs in the faeces of II people; 2 of the cases were Studies made by ROMIG(1980) and ROMIG, LUCIUS & established infections, 4 were spurious (eggs only found FRANK (1980) on the «brain-worms» of both D. hospes once) and the other 5 people were examined only once. and D. dendriticum showed differences in location, REINTHALER et at. (1988) examined 479 stool specimens number, size and behaviour. D. hospes metacercariae are and found Dicrocoelium eggs in 0,4%. preferentially located dorsally on the antennary lobes of Other genuine human infections reported from Africa, the deutocerebrum, their number is usually 2 with a ma- such as in northern Africa, Lebanon, Syria and Egypt do ximum of 4, their size is 410-450 urn, and their effects in not concern H. hospes but D. dendriticum. modifying the behaviour of the ant is not related to tem- perature, whereas those of D. dendriticum are mainly lo- cated ventrally on suboesophagic ganglia and sometimes Geographical distribution on the optic lobes, their number is usually I with a maxi- In the Ethiopian region, the species D. dendriticum se- mum of 3, their size is 320-410 urn, and their effects in ems to be replaced by D. hospes, the dicrocoeliid species modifying the behaviour of the ant are related to low en- present in almost all west African countries. D. hospes has vironmental temperature. been recorded so far from Angola (GRABER & PERROTIN, 1983), Cameroon (GRABER & OUMATIE, 1964), Central First intermediate hosts African Republic (GRABER & OUMATIE, 1964; GRABER & PERROTIN, 1983), Chad (GRABER & OUMATLE, 1964), In the first report, BOURGAT, SEGUI & BA YSSADE- Congo (GRABER & PERROTI , 1983), Etiopia (GRABER & DUFOUR (J 975) demonstrated that the snail hosts of D. PERROTI , 1983), Ghana (ODEI, 1966; WOLFE, 1966), hospes in Togo are species of the terrestrial genus Limi- Guinea Republic (GRABER & OUMATIE, 1964; KAJUBIRI colaria (). Late studies by BOURGAT & & HOHORST, 1977), Kenya (CHUNGE & DESAI, 1989), KULO(1979) and CHEVALLIER (1979) identify the spe- Mali (MALEK, 1980; TEMBELY et aI., 1988), Niger (TA- cies as aurora (Jay, 1839) and L. bourgati GER-KAGAN, 1979), Nigeria (ROCHE, 1948, identified in Chevallier, 1979. Limicolaria flammea, L. felina and the report as D. dendriticum; HARMON & OYERINDE, another species similar to L. kambeul were found to play 1976; SCHILLHORN VAN VEEN et al., 1980; OGU RI ADE the role of first intermediate host of D. hospes in the & ADEGOKE, 1982; ADEOYE & FASHUYI, 1986; FASHUYI Ivory Coast (LUCIUS, ROMIG & FRA K, 1980). More re- & ADEOYE, 1986; REINTHALER, MASCHER, KLEM & cently, FASHUYI & ADEOYE (1986) experimentally ex- SIXL, 1988), Senegal (VASSILIADES, 1978; MALEK, 1980; posed the snail species Limicolaria flammea, L. stria- DIAW, 1982, 1988), Sierra Leone (KI G, 1971; WI- tula, an unidentified Limicolaria, and Achatina sp. LLIAMS, 1969; ASANJI & WILLIAMS, 1984, 1987), Sudan collected in Nigeria, and laboratory-reared Lamellaxis (Looss, 1907; although it was not found in extensive sur- gracilis, to fully embryonated eggs of D. hospes for 48 veys made by MALEK, 1959), Tanzania (MAHLAU, 1970; h. Larval stages develop to maturity in Limicolariaflam- GRABER & PERROTIN, 1983), Togo (BOURGAT, SEGUIN & mea, L. striatula and Lamellaxis gracilis, rates of infec- BAYSSADE-DuFOUR, 1975; BOURGAT & KULO, 1979; tion being 30, 20 and 8%, respectively. None of the other CHEVALLIER, 1979; GNINOFOU, 1988), Uganda (ODEI, snails became infected. 1966; KAJUBIRI & HOHORST, 1977; THURSTON, 1970, 1972), Zaire (VA DEN BERGHE & DENECKE, 1938), and Second intermediate hosts Zambia (GRABER & PERROTIN, 1983). The African distri- bution of this parasite between latitudes of 20° Nand 20° Formicidae ants act as second intermediate hosts. The S has been related to the distribution of its intermediate metacercariae of D. hospes have been found in two ant snail hosts (KAJUBIRI & HOHORST, 1977). species in Togo: Dorylus sp. and Crematogaster sp. 194 S. MAs-CoMA & M.D. BARGUES

(BOURGAT, SEGUIN & BA YSSADE-DuFOUR, 1975). Ex- much damage, even in heavy infections (DIA W, 1982). perimental infections gave positive results in several ant Up to 973 adults were found in a cattle liver (TAGER-KA- species from the Ivory Coast, such as Camponotus com- GAN, 1979). pressiscapus, C. chrysurus, C. vividus and C. acvapi- mensis, although mature metacercariae infective for de- Diagnosis finitive hosts were only obtained in C. compressiscapus because of ant-rearing difficulties (LUCIUS, ROMIG & Certain diagnosis is only made by detection of eggs in FRANK, 1980). faeces over a long period without having ingested infec- ted animal liver. Serological tests, such as immunodiffu- sion and ELlSA, present the problem of cross-reactions Epidemiology (common antigens) with Fasciola gigantica and Schisto- Studies carried out at slaughterhouses in different en- soma bovis, two digeneans existing in both domestic ani- demic African countries demonstrate that, when present, mals and man in the same African countries as D. hospes this dicrocoeliid usually reaches high prevalences in cat- (FAGBREMI & OBARISIAGBON, 1991). tle: 58,5% in Ghana (ODEI, 1966), 80,6% in Uganda (KAJUBIRI & HOHORST, 1977). Treatment Prevalences in African domestic animals differ accor- ding to regions, season and definitive host species. In cat- The several drugs having shown to be effective against tle the following high prevalences were found: 0,0-54,0- D. dendriticum may similarly be applied against D. hos- 94,0% in different regions of Niger, 80,6% in Uganda, pes infection. 71 ,0% in Tanzania, 61,8% in Sierra Leone, 58,8% in Ghana, and 57,8% in Chad (TAGER-KAGAN, 1979; KAJU- Prevention and control BIRI & HOHORST, 1977; ASA JI & WILLlAMS, 1984, 1987; ODEI, 1986; GRABER & OUMATIE, 1964; SCHILL- Preventive and control measures already described to HORN VA VEE et aI., 1980). Lower prevalences found be useful in dicrocoeliasis by D. dendriticum can be ap- in cattle were 40,0% in Togo (BOURGAT, SEGUIN & plied in a similar way against D. hospes. BAYSSADE-DuFOUR, 1975), 18,5% and 45,0% in Nigeria (ADEOYE & FASHUYI, 1986; OBIAMIWE, 1986), 3,0- 38,0% in Senegal (MALEK, 1980; DIAw, 1982, 1987), and EURYTREMA PANCREATlCUM 16,6% in Mali (MALEK, 1980). Zebus appear to be more susceptible to infection (12,0%) than N'dama cattle Morphology (6,7%) (DIAw, 1982), and bovine females more suscepti- ble than males (ASANJI & WlLLlAMS, 1987). The adult stage is thick, 8-16/5-8 mm in size, with a Sheep and goats present lower prevalences: 2,0-14,0- tegument provided of caducuous spines. The oral sucker 27,% in sheep and 10,0-15,0-26,0% in goats in different is subterminal and is larger than the acetabulum. The regions of Niger (TAGER-KAGAN, 1979), 0,97% and pharynx is small, the oesophagus short and the caeca do 3,33% respectively in Senegal (DIAw, 1987), 3,2% and not reach the posterior extremity of the body. The testes 0,0% respectively in Sierra Leone (ASANJI & WI- are lobed, symmetrical, intercaecal, and immediately LLIAMS, 1987). postacetabular. The long and slender cirrus pouch ex- Different relationships of prevalences to the season tends from the anterior margin of the acetabulum to al- have been found. Lower prevalences in cattle were found most the intestinal bifurcation. The ovary is relatively in the dry season in Togo (TAGER-KAGAN, 1979) and Ni- small, lobed, postesticular, submedian and is located in geria (SCHlLLHORN V AN VEEN et aI., 1980; ADEOYE & the middle third of the body. The vitelline follicles are FASHUYI, 1986), whereas the opposite, that is, the higher disposed in two postesticular lateral fields mostly overl- prevalence in the dry season were detected in Sierra Le- ying the caeca. The uterus is markedly coiled, fills the one, Ghana and Tanzania (ASANJI & WILLlAMS, 1984). posterior part of the body, and goes up to finish in a di- rectly postbifurcal, median, ventral, genital pore. The embryonated eggs, 40-50/23-24 I1m in size, are indistin- Pathology, symptomatology guishable from those of D. dendriticum. and clinical manifestations

Concerning humans, ROCHE(1948) showed a median Location and definitive hosts longitudinal section through the worm in the section of the human liver; the only pathological change was a mo- This species is normally found within the pancreatic derate degree of fibrous thickening of the portal tract. ducts and less frequently in the bile ducts. Natural defi- IQ G(1971) reported, in two heavily infected patients, nitive hosts are cattle, sheep, goats, rabbits, hogs, water hepatitis-like symptoms: one of them had jaundice, both buffaloes, camels (Came/us bactrianusi, monkeys (Ma- had raised bilirubin and transaminase levels. caca syrichta) and carabao. Experimentally, this dige- Concerning animals, D. hospes does not seem to cause nean can be developed in additional definitive hosts. Human liver flukes: a review 195

Reports in humans snails. The cercaria contained in the daughter sporocysts has a short stumpy tail, and the structure of its body is According to TANG & TANG (1977), E. pancreaticum very similar to that of other dicrocoeliid cercariae (TANG, has so far been reported in man once each in Hong Kong 1950). In experimentally-infected snails, a great number and in Jiang-su Province, China and in at least six cases of daughter sporocysts are found on the outer wall of the in Japan. More recently, ISHll et al. (1983) reported a hu- posteriormost part of the stomach. Soon after being shed, man infection in a 70-year-old Japanese woman with daughter sporocysts are found near mother sporocysts in gastric cancer in Fukuola Prefecture, Japan, who harbou- the subrenal and visceral sinuses. Daughter sporocysts red 15 adult E. pancreaticum in the dilated pancreatic migrate towards the mantle collar through the rectal sinus ducts at autopsy. The number of eosinophils in the blood (if originally parasitic in the visceral sinus) or the lateral was within normal limits. Thus, parasites could be mi- sinus (if parasitic in the subrenal sinus).Daughter spo- croscopically determined: body 10-1 1/5-7 mm; oral suc- rocysts then penetrate the mantle to the exterior. Emer- ker 2,0-2,111,9-2,0 mm; ventral sucker 1,4-1,6/1 ,5-1,6 gent sporocysts are spindle-shaped, whereas those migra- mm; embryonated eggs in the uterus 43,2-50,9/27,6- ting through the sinuses are of elongate fusiform shape. 33,0 urn. The same year, TAKAOKA et al. (1983) repor- The process of emergence takes 5-10 minutes (KOZUT- ted another human case of a 57-year-old female farmer SUMI & ITAGAKI, 1989). from Notsuharu, Oita, Japan, who was admitted to hos- When sporocysts from the snails are fed to grasshop- pital complaining of hypochondralgia which had lasted 3 pers, the contained cercariae penetrate the gut and encyst months. She was given a pancreatectomy. Histopatholo- in the hemocoel. Metacercarial cysts grow to maximum gical examination of the pancreas reavealed 3 E. pancre- size in about 9 days and mature in 2 additional weeks. aticum in the dilated pancreatic duct. When grasshoppers are fed to goats, they develop to According to MORIYAMA (1982 b), all human cases of young worms in the pancreas (BASCH, 1965). eurytremiasis seem to be due to E. pancreaticum, at least In experimentally-infected rabbits, the trematodes in Japan. were found mainly in the duodenum before migrating to the pancreas through the pancreatic duct. In the pan- creas, the trematodes reached the adult stage in 45 days Geographical distribution and on day 75-80 they began to lay eggs (PANIN& This species is known to occur in many areas of eas- KSEMBAEVA, 1971). Infected grasshoppers, when fed to tern Asia, such as the old USSR (KSEMBAEVA, 1967: in rabbits, lead to the development of juvenile flukes in 30 south-eastern Kazakhstan; LOGACHEVA, 1974: in Kirgi- days and fully mature flukes in 90 days, the prepatent zia; DVORYADKIN, 1969: Amur region of the Far Eastern period being about 100 days. (CHINONE & ITAGAKI, USSR; NADIKTO & ROMANENKO, 1969: Primorsk Terri- 1976). In experimentally-infected lambs, the develop- tory of Far-Eastern Russia), Korea (JANG, 1969), Japan ment to adults takes 87-89 days (DVORYADKIN, 1969). (CHINO E, MARUY AMA & ITAGAKI, 1976; CHINONE & ITAGAKJ, 1976; TANG & TA G, 1977; ISH" et al., 1983; First intermediate hosts TAKAOKA et al., 1983), Mongolia (Gu et al., 1990), southern and northern China (TANG, 1950; TANG & Different terrestrial snails, mainly of the family Brady- TANG, 1977; TANG et al., 1979), Hong-Kong (TANG & baenidae (), are used as first intermediate host TANG, 1977), Vietnam (NGUEN TKI LE & MATEKIN, by E. pancreaticum, depending of the geographical zone. 1978), Malaysia (BASCH, 1965), Philippines (EDUARDO, The following species have been reported with natural MANUEL & TONGSON, 1976), and also Mauritius Island and/or experimental infection by the larval stages: in in the Indian Ocean, near Madagascar (LE Roux & south-eastern Kazakhstan, Bradybaena lantzi and Cat- DAR E, 1955). Ascriptions to this species in South Ame- haica plectotropis (KSEMBAEVA, 1967); in the Amur re- rica do not seem in fact to be E. pancreaticum, but E. eo- gion of the Far Eastern USSR, Bradybaena arcasiana elomaticum (Giard et Billet, 1892) Looss, 1907 (TRA- and B. dieckmanni (DVORY ADKI , 1969); in the Primorsk VASSOS, TEIXEIRA DE FREITAS & KOHN, 1969), a Territory of Far-Eastern Russia, Bradybaena fragilis, B. proximal species also present in eastern Asia and which selskii, B. middendorffi, B. maacki and B. arcasiana has also been differentiated karyologically from E. pan- (NADYKTO, 1973); in Kirgizia, Jaminia albiplicata, creaticum in areas where both species overlap, such as in Bradybaena plectotropis, B. phaeozona and B. lantri Japan (MORIY AMA, I 982a). (LOGACHEVA, 1974); in Korea, Acusta despecta (JAG, 1969); in Mongolia, Ganesella virgo (Gu et al., 1990); in China, Bradybaena similaris and Cathaica ravida sie- Life cycle boldtiana (TANG, 1950), Ganesella stearnsii, G. japo- The egg and the miracidium are very similar to those nica and G. myomphala (TANG & TANG, 1977), and G. described for other dicrocoeliids. The eggs hatch only af- virgo (TANG et al., 1979); in Japan, Fruticicola siebol- ter being eaten by the snails. The daughter sporocysts de- diana and B. similaris stimpsoni (MIYATA, 1965), and B. velop within the mother sporocyst into large complicated similaris (CHLNONE, MARUYAMA & ITAGAKI, 1976; Ko- sacs with a heavy outer wall, and they escape from the ZUTSUMI & ITAGAKI, 1989); and in Malaysia B. similaris 196 S. MAs-CoMA & M.D. BARGUES

(BASCH, 1965). A land snail (Macrochlamys) has been described, eosinophilia being within normal limits (TA- indicated as probable first intermediate host in Mauritius KAOKA et al., 1983; ISHII et al., 1983). (LE Roux & DAR E, 1955). In animals, few changes are observed in the pancreas in light infections. In moderate and heavy infections, ca- tarrh, ectasis, hyperplasia or fibrosis of the pancreatic Second intermediate hosts duct are found (SHIEN et al., 1979).Eggs may penetrate The second intermediate hosts are mainly grasshop- into the walls of ducts causing inflammatory foci and pers. Metacercariae develop and encyst in the abdominal granulomata in which plasma cells and eosinophils pre- cavity of the following species: Conocephalus macula- dominate (BASCH, 1966). Ius in Korea (lA G, 1969), Malaysia (BASCH, 1965), and Japan (CHINONE & ITAGAKI, 1976); C. chinensis in Diagnosis China (TANG et al., 1979), the Amur region (DVORY AD- KIN, 1969), the Primorsk Territory ( ADYKTO, 1973), Human cases have been diagnosed at a hispathological and Mongolia (Gu et al., 1990); C. gladiatus in Korea examination after pancreatectomy (TAKAOKA et al., (lAG, 1969); C. dorsalis and C. discolor in Kirgizia 1983) or at autopsy (lSHII et al., 1983). Egg finding in (LOGACHEVA, 1974); C. percaudatus in the Amur region coprological techniques presents the problem of confu- (DVORY ADKIN, 1969); C. fuscus and Platycleis interme- sion with D. dendriticum eggs, from which they are in- dia in south-eastern Kazakhstan (KSEMBAEVA, 1967); distinguishable. Although they are also indistinguishable and Oecanthus longicaudus and Epocromia sp. in the from D. hospes eggs, in this case the different geograp- Primorsk Territory (NADIKTO & ROM A E KO, 1969; hical distribution (D. hospes is confined to the African ADYKTO, 1973). The ant (Technomyrmax) has been in- continent) becomes helpful. dicated as probable second intermediate host in Mauri- tius (LE Roux & DARNE, 1955). Treatment

Numerous have been unsuccessfully as- Epidemiology sayed in animals.Finally, LI et al. (1983) demonstrated Temperature has a noticeable effect on the develop- that praziquantel (oral doses of 50-70 rng/kg) was very ment of the parasite in the snail. In China, development effective for the treatment of heavy infection in sheep in snails stops under 10° C (TANG et al., 1979). In Japan, (more effective than hexachloroparaxylene). at 26° C the development of the larval stages is accelera- ted, cercariae developing in 81 days. The length of the Prevention and control development depends on the year period (shortest in March). Mature daughter sporocysts showed a 2-3 day The prevention and control measures are similar to shedding rhythm from snails (CHINO E, MARUY AMA & those applied for dicrocoeliasis, with the only difference ITAGAKI,1976). that in eurytrerniasis the second intermediate hosts are Under field conditions in the Amur region of the Far grasshoppers instead of ants. Eastern USSR, eggs remained viable from April to No- vember but could not survive the cold winter. In snails, the development to cercariae took 12 to 13 months at tem- AMPHIMERUS PSEUDOFELlNEUS peratures varying from 8 to 25° C(DVORYADKIN, 1969). In Korea, snails are infected with the eggs in autumn, and The species Opisthorchis guayaquilensis Rodriguez, the miracidia become mother sporocysts in the liver du- G6mez et Montalvan, 1949 was originally described from ring the winter. Daughter sporocysts originated in the a human in rural Ecuador by RODRIGUEZ, GOMEZ & spring penetrate the membrane of the mantle cavity in MO TALVAN (1949). This species was later transferred to June to July.They are eaten by grasshoppers in summer the genus Amphimerus Barker, 1911 of the same family and autumn and encyst in the abdominal cavity (lA G, Opisthorchiidae (ARTIGAS & PEREZ, 1964; THATCHER, 1969). In the Primorsk Territory, in snails the second spo- 1970) and synonyrnized with the species Amphimerus rocyst generation produces cercariae 10-11 months after pseudofelineus (Ward, 190 I) Barker, 1911 (ARTIGAS & infection. In grosshoppers, metacercariae become infec- PEREZ, 1964). THATCHER (1970) did not agree with this tive in 23-40 days. In the final host (sheep and cattle) fully synonymy and considered A. guayaquilensis distinct from embryonated eggs appear in faeces 80-100 days later. The A peudofelineus because of the extent of the vitellaria: in complete life cycle takes 500-560 days (NADYKTO, 1973). A. guayaquilensis the vitelline follicles extend to near the posterior end of the body, well beyond the posterior testis, whereas in A peudofelineus the vitellaria reach the poste- Pathology, symptomatology rior testis but do not extend beyond it. Later on, GOMES and clinical manifestations (1977) had the opportunity to study the morphometries of In humans, complaints of hypochondralgia lasting 3 various Amphimerus species in Brazil and again conclu- months and dilation of the pancreatic ducts have been ded on the validity of the above mentioned synonymy. Human liver flukes: a review 197

A. pseudofelineus has apparently a wide geographic numerous areas of Europe (Russia, Germany, Hungary, range and low specificity for its definitive host. It is a pa- France, Italy, etc.), as well as from North America (UL- rasite of the bile-ducts of dogs, coyotes, domestic cats, MER, 1975). Eggs measure 27-35/12-16 urn, Metacerca- and marsupials (Didelphis marsupialis, Philander opos- riae develop in fish (Y AMAGUTI, 1971). The determina- sum) in the U.S.A., Panama, Colombia, Venezuela, tion of the metacercariae found encysted in cyprinoid Ecuador, and Brazil (CABALLERO, GROCOIT & ZERE- fishes by SCHUURMA S-STEKHOVE (1931) as belon- CERO, 1953; CALERO, ORTIZ & DE SOUZA, 1955; THAT- ging to P. truncatum is doubtful according to Y AMA- CHER, 1970; MAYAUDO , 196911970; TODD, BERGE- GUTI (1975). LAND & HICKMAN, 1975; GOMES, 1977). Its life cycle Instances of human infection are relatively few (BITT- does not seem to be elucidated so far, but it probably fo- NER 1928; PETROV, 1940; DELlANOVA, 1957). Appa- llows the same pattern as other Amphimerus species, rently, however, human prevalences may be relevant in with aquatic snails shedding cercariae and fish harbou- given zones, as in the Alekseev District of Tataria (old ring encysted metacercariae (FONT, 1991). USSR), a district situated near the River Kama, where P. RODRIGUEZ, GOMEZ & Mo TALVAN (1949) made fae- truncatum was found in the bile of up to 31 patients cal examinations of 245 persons in a lowland area of (KHAMIDULLI et al., 1991). VI OGRADOV (1892) repor- Ecuador and found 18 of them passing opisthorchiid ted young stages of a fluke (later presumed to have been eggs. They also found several dogs in the same area pas- P. truncatumy from a human source in Russia (ULMER, sing similar eggs, and upon sacrificing the dogs they 1975). Both eggs and metacercariae of P. truncatum can found the worms which they describe as O. guayaquilen- be distinguished from those of Opisthorchis felineus (ZA- sis. They believed it was the eggs of this species that had BLOTSKll, 1973). There is no study on pathology at hu- been seen in human faeces. The eggs of this species ob- man level, but liver pathological effects are known in se- tained from humans in Ecuador measured 27-35/11- als (Porov, KOROLEV & SKOROKHOD, 1985). 17 urn (average 31,5/l3,5 urn), No additional human re- port of this species appears in the literature. Worth mentioning is, however, that in Colombia, RESTREPO METORCHIS CONJUNCTUS (1962) reported finding opisthorchiid-like eggs in the fae- ces of 6 out of 176 persons residing on the Amazon river. First described from the bile duct of an American red fox in the London zoological gardens, it was later obser- ved that this species occurs in orth America, especially AMPHIMERUS NOVERCA in Canada, where it has a wide distribution. It has been also reported from several areas in the U.s.A. (Wiscon- Species originally described within the genus Opist- sin, Minnesota, Maine, New York). horchis Blanchard, 1895 (0. noverca Braun, 1902; syn.: The first record of M. conjunctus in man was in the Distoma conjunctum Lewis et Cunningham, 1872 nee stool from an Indian patient in Saskatchewan, Canada Cobbold, 1859 - see NEVEU-LEMAIRE, 1936) and later (CAMERO , 1945). Eggs of this species have also been transferred to the genus Amphimerus Barker, 1911 (see reported in stools of a human native of Greenland (BAB- Y AMAGUTI, 1971), its adults do not appear to be location BOIT, FRYE & GORDON, 1961). specific, since they have been recovered from the lung It is a parasite of the bile ducts of the dog, wolf, cat, and pancreatic ducts of stray dogs (SAHAI, 1969), from fox, raccoon, mink, and fisher (Y AMAGUTI, 1971; DICK the bile ducts of pigs (BHALERAO, 1931) and also in pan- & LEONARD, 1979; WOBESER, RUNGE & STEWART, creatic ducts of pigs in India (SIHA, 1968). Adults are 1983). Pancreatic involvement has also been detected in 9,5-12,7/2,5 mm in size and the eggs measure 34/19 urn. wolves (WOBESER, Ru GE & STEWART, 1983). The This species has been reported in the gall bladder at worm measures 1,0-6,6/0,59-2,6 mm in length/width, autopsy of two Mohammedans in India (MC Co ELL, and its small eggs measure 22-32/11-18 urn. A detailed 1876, 1878). These reports offer considerable doubt con- morphometric study of the adult stage was carried out by cerning the specific determination of the human flukes WATSON (1981). (LEIPER, 1913). Proliferative changes in pancreatic duct The life cycle is similar to that of species of Clonor- epithelium and some epithelial desquamation and peri- chis and Opisthorchis. It has been experimentally reared ductal fibrosis have been observed in pigs, in cases of in dogs, cats, red and silver foxes, minks, ferrets, racco- heavy infections in the pancreas (SI HA, 1968). ons, and cotton rats. The life span of the adult stage may exceed 5 years (CAMERON, 1944). The aquatic hydrobiid snail Amnicola limosa porata serves as the first interme- PSEUDAMPHISTOMUM TRUNCATUM diate host and the common sucker, Catostomus commer- sonii, as the fish second intermediate host, in which the P. truncatum parasitizes the bile ducts of various infective encysted metacercariae are found in the mus- mammals including cat, dog, fox, seal, skunk, mink, ot- cles (CAMERO ,1944). ter, etc. (Y AMAGUTI, 1971). Adults are small digeneans In Canada, sledge dogs appear to be commonly infec- (1,64-2,5/0,6-1,0 mm) which have been reported from ted, and heavy infections may lead to death. At the level 198 S. MAS-COMA & M.D. BARGUES of the bile ducts, adult worms may cause lesions similar tinal trematode diseases.In: Tropical Medicine and Parasito- to those produced by Clonorchis sinensis and Opisthor- logy (R.Goldsmith & D. 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schen Gesellschaft fiir Parasitologie e.V., 26-29 Marz 1980, Chinese Journal of Veterinary Science and Technology, 3: 15- Giessen. Praktische Tierarzt, 61 (4): 354. 16. (in Chinese) ROMIG(T.), LuClUS(R.) & FRA K (W.), 1980.- Cerebral larvae in ISHII (Y.), KOGA (M.), FUJlNO(T.), HIGO (H.), ISHIBASHI(J.), aKA the second intermediate host of Dicrocoelium dendriticum (Ru- (K.) & SAITO (S.), 1983.- Human infection with the pancreas dolphi, 1819) and Dicrocoelium hospes Looss, 1907 (Trema- fluke, Eurytrema pancreaticum. American Journal of Tropical toda, Dicrocoeliidae). Zeitschrift fiir Parasitenkunde, 63: 277- Medicine and Hygiene, 32: 1019-1022. 286. JANG(D.H.), 1969.- [Study on Eurytrema pancreaticum. 11. Life SCHILLHORNVAN VEEN (T.W.), FOLARANMI (P.a. B.), USMAN(S.) cycle]. 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Sbornik Nauchnykh Trudov dendriticum in man and monkeys in the Belgian Congo. Annals Kirgizskogo Gosudarstvennogo Meditsinskogo lnstituta (Vo- de la Societe Beige de Medecine Tropicale, 18: 509. prosy biologii iporatitologii v Kirgizii}, 95: 31-34. (in Russian) V ASSILlADES(G.), 1978.- Capacite de resistence it la secheresse de MIYATA(I.), 1965.- The development of Eurytrema pancreaticum la limnee (Lymnaea natalensisi rnollusque hote intermediaire de and Eurytrema coelomaticum in the intermediate host snails. In: Fasciola gigantica au Senegal. Revue d'Elevage et de Medecine Progress of Medical Parasitology in Japan, Volume 2 (K. Mo- WTerinaire des Pays Tropicaux, 31: 57-62. rishita, Y. Komiya & H. Matsubayashi edit.), Meguro Parasito- WILLlAMS (M.O.), 1969.- First record of dicrocoeliasis in cattle in logical Museum, Tokyo: 347-357. Sierra Leone. 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TAG(C.), CUI (G.), Do G (Y.), WA G (Y.), NULlMAJABU, Lu Transactions of the Royal Society of Tropical Medicine and Hy- (H.), ZHA G (C), CHE(M.), Su (G.) & QUIA (Y.), 1979.- giene (1912-1913), 6: 265-297. [Studies on the biology and epidemiology of Eurytrema pancre- Me CO ELL (l.F.P.), 1876.- On the Distoma conjunctum as a hu- aticum (lanson, 1889) in Heilungkiang Province]. Acta Zoolo- man entozoon. Lancet, I: 343-344. gica Sinica, 25: 234-242. (in Chinese) Mc CO ELL (J.F.P.), 1878.- Distoma conjunctum. Lancet, I: 476. TA 'G (C.C.), 1950.- Studies on the life history of Eurytrema pan- EVEU-LEMAIRE(M.), 1936.- Traite d'Helminthologie Medicate creaticum Janson 1889. Journal of Parasitology, 36 (Sect. I): et Yeterinaire. Vigot Freres, Editeurs, Paris, 1514 pp. 559-573. SAHAI (B.N.), 1969.- A survey of the helminth parasites of stray TA G (Z.) & TA G (C.), 1977.- [The biology and epidemiology of dogs around Bareilly, Uttar Pradesh. Indian Veterinary Journal, Eurytrema coelomaticum (Giard et Billet, 1892) and Eurytrema 46: 734. pancreaticum (Janson, 1889) in cattle and sheep in China]. Acta SINHA(B.K.), 1968.- Opisthorchis noverca Braun 1902 in the pan- Zoologica Sinica, 23: 267-282. (in Chinese) creas of domestic pig (Sus scrofa domestica) in Bihar. Curl'. Tsxvxssos (L.), TEIXEIRA DE FREITAS(l.F.) & KOH (A.), 1969.- Sci., 37: 22-23. Trematodeos do Brazil. Memorias do lnstituto Oswaldo Cruz; Y AMAGUTI (S.), 1971.- Synopsis of Digenetic Trematodes of Ver- 67: 1-886. tebrates. Vol. [and IT. Keigaku Publishing Co., Tokyo, 1074 pp. + 349 pI.

Amphimerus pseudofelineus Pseudamphistomum truncatum ARTIGAS (P. de T.) & PEREZ(M.D.), 1964.- Consideracoes sabre Opisthorchis pricei Foster, 1939, 0. guayaquilensis Rodrfguez, BITTNER (H.) & SPREHN(C.E.W.), 1928.- Trematodes. Saugwur- G6mez et Montalvan 1949, e O. pseudofelineus Ward 190 I. mer. BioLogie der Tiere Deutschlands, 27 (5): 1-133. Descricao de Amphimerus pseudofelineus minutus n. subsp. Me- DELlANOVA (R.S.), 1957.- [Distribution of helminthiasis of dogs morias do lnstituto de Butantan, 30: 157-166. in different geographical zones of SSSR I. Doklady Academii CABALLERO (E.), GROCOTT (R.G.) & ZERECERO(M.C.), 1953.- Nauk Urbek SSR, 10: 65-71. (in Russian) Helmintos de la Republica de Panama. IV. Redescripci6n de al- KHAMIDULLlN (R.l.), LYUBINA (V.S.), KHAMIDULLlN(I.R.) & ME- gunas forrnas de trematodos ya conocidos y descripci6n de una DINSKII (B.L.), 1991.- [Trematode infections in Tataria]. Medit- nueva especie de Amphimerus. Anales del lnstituto de Biologia, sinskaya Pararitologiya i Parazitarnye Bolezni, 2: 60-61. (in Mexico, 23: 181-201. Russian) CALERO(M.), ORTlZ (P.O.) & DE SOUZA (L.), 1955.- Trernatodia- PETROV (A.M.), 1940.- [Parasitic worms of Mustelidae of the sis en gatos de Arraijan y Chorrera (Rep. de Panama). Archivos Moscow zoological gardens]. Trudy Moskovsk. Zooparka, I: de Medicina Panameiios, 4: 37-41. 202-213. (in Russian) FONT(W.F.), 1991.- Life cycle of Amphimerus elongatus (Trema- Porov (V. .), KOROLEV (V.A.) & SKOROKHOD(L.A.). 1985.- toda: Opisthorchiidae). Journal of the Helminthological Sociery [Pseudall7phistoll7ulI7 infection in Pusa caspica]. Vestnik Zoolo- of Washington, 58: 24-30. gii, 2: 51-54. (in Russian) GOMES (D.C.), 1977.- Contribuciio ao conhecimenio dos helmin- SCHUURMAS-STEKHOVE (J.H.Jr.), 1931.- Der zweite Zwischen- tos parasitos de marsupiais no Brasil da coleciio helmintologica win von Pseudamphistomum truncatum (Rud.) nebst Beobach- do lnstituto Oswaldo Cruz; Thesis, Universidade Federal Rural tungen uber andere Trematoden-Larven. Zeitschrift fiir Parasi- do Rio de Janeiro, Rio de Janeiro, vi + 102 pp. tenkunde, 3: 747-764. MAYAUDON(T.H.), 1969/1970.- Contribuci6n al estudio de la ULMER (M.J.), 1975.- Other trematode infections. In: Diseases fauna parasitaria de los mamfferos de Venezuela. I.Nuevo tre- transmitted from animals to man. 6th Edition (W.T. Hubbert, matode para Venezuela Amphimerus pseudofelineus (Word, W.F. McCulloch & P.R. Schnurrenberger edit.), C.C. Thomas 190 I) parasite del gato (FeLis felis (catus) domesticusi.Revista Publ., Springfield-lllinois: 646-677. de Medicina veterinaria y Parasitologia, Maracay- Venezuela, VINOGRADOV(K.N.), 1892.- On a new species of distome tDisto- 23 (1/8): 315-316. mum sibiricum) in the human liver. lzvest. Imp. Tomsk Univ., 4: RESTREPO(.M.), 1962.- Estudio parasitol6gico de una regi6n del 116-160. (in Russian) Amazonas Coombiano. Antioquia Med., 12: 462-484. Y AMAGUTI (S.), 1971.- Synopsis of Digenetic Trematodes of Ver- RODRIGUEZ(l.D.), GOMEZ (L.F.) & MONTALvAN (J.A.), 1949.- El tebrates. Vol. J and [I. Keigaku Publishing Co., Tokyo, 1074 pp. Opisthorchis guayaquilensis una nueva especie de Opisthorchis + 349 pI. encontrada en el Ecuador. Revista Ecuatoriana de Higiene y Y AMAGUTI (S.), 1975.- A Synoptical Review of Life Histories of Medicina Tropical, 6: 11-24. Digenetic Trematodes of Vertebrates. Keigaku Publishing Co., THATCHER (V.E.), 1970.- The genus Amphimerus Barker, 1911 Tokyo, 590 + 219 pI. (Trematoda: Opisthorchiidae) in Colombia with the description ZABLOTSKII (V. I.), 1973.- [Differential diagnosis of eggs and met- of a new species. Proceedings of the Helminthological Society cacercariae of Opisthorchis felineus and Pseudamphistomum of Washington, 37: 207-211. trtlllcatllll7]. Meditrinskaya Parazitologiya i Paraiitarnye Bo- TODD (K.S.Jr.), BERGELA D (M.E.) & HICKMAN (G.R.), 1975.- Iezni, 42: 334-338. (in Russian) Amphimerus pseudofelineus infection in a cat. Journal of the American Veterinary Medical Association, 166: 458-459. Metorchis COlYUIlCtllS ULMER (M.J.), 1975.- Other trematode infections. In: Diseases transmitted from animals to man. 6th Edition (W.T. Hubbert, BABBOTT(F.L. Jr.), FRYE(W.W.) & GORDON(J.E.), 1961.- lntesti- W.F. McCulloch & P.R. Schnurrenberger edit.), c.c. Thomas nal parasites of man in Arctic Greenland. American Journal of Publ., Springfield-Illinois: 646-677. Tropical Medecine and Hygiene, 10: 185-190. CAMERO (T.W.M.), 1944.- The morphology. and life history of Metorchis conjunctus (Cobbold, 1860). Canadian Amphimerus noverca Journal of Research, Section D, Zoological Sciences, 22: 6-16. BHALERAO(G.D.), 1931.- Trematode parasites of pigs in Bengal. CAMERO (T.W.M.), 1945.- Fish-carried parasites in Canada. Ca- Records of the Indian Museum, 33: 475. nadian Journal of Comparative Medicine, 9: 245-254; 283-286; LEIPER (R.T.), 1913.- Observations on certain helminths of man. 302-311. 218 S. MAS-COMA & M.D. BARGUES

DICK (T.A.) & LEO ARD(R.D.), 1979.- Helminth parasites of fis- 1860) Looss, 1899 (Trematoda: Opisthorchiidae). Proceedings of her Manes pennanti (Erxleben) from Manitoba, Canada. Jour- the Helminthological Society of Washington, 48: 172-176. nal of Wildlife Diseases, 15: 409-412. WOBESER (G.), Ru GE (W.) & STEWART (R.R.), 1983.- Metorchis MILLS (lH.L.) & HIRTH (R.S.), 1968.- Lesions caused by the he- conjunctus (Cobbold, 1860) infection in wolves (Canis lupus), patic trematode, Metorchis conjunctus Cobbold, 1860. A com- with pancreatic involvement in two animals. Journal of Wildlife parative study in Carnivora. Journal of Small Animal Practice. Diseases, 19: 353-356. 9: 1-6. Y AMAGUTI(S.), 1971.- Synopsis of Digenetic Trematodes of Ver- WATSO (T.G.), 1981.- Evaluation of actual and relative measure- tebrates. Vol. I and 11.Keigaku Publishing Co., Tokyo, 1074 pp. ments used in the description of Metorchis conjunctus (Cobbold. + 349 pI.