by Vyacheslav MORDVINOV, Dr. Sc. (Biol.), Deputy Director for Science, Institute of Cytology and Genetics, RAS Siberian Branch (Novosibirsk), Dagmara FURMAN, Senior Researcher, Laboratory of Molecular and Genetic Systems of the same institute

There is an ever growing body of evidence on the tight link between helminthic invasions and malignancies in organs infested with helminths. Here in Russia the worst danger comes from the liver fluke ("sucker") Opisthorchis felineus (cat liver fluke), also known as the "Siberian opisthorchis", a hepatic parasite causing opisthorchiasis, or "the Ob disease". Its diagnostics still leaves much to be desired, while the available medication entails unwanted side-effects. Further research is needed into the biology of the pathogen and the pathology it causes, and so is elaboration of adequate identification and treatment strategies. The Institute of Cytology and Genetics (RAS Siberian Branch) is realizing a major project on all-round studies of O. felineus. New facts, important both in overall biological and in medical aspects, have been collected.

WHAT WE KNOW ABOUT CAT LIVER FLUKES

Helminthiasis (helminthic invasion*) affects or else threatens a vast number of people. In keeping with the classification of the International Agency of Research for Cancer (IARC), two helminthic species-the liver flukes Clonorchis sinensis and Opisthorchis viverrini- were ranked still back in 1994 among the number one group of deadly cancerogens. The former occurs in China, Korea, Japan, Laos, Viet Nam and Russia's Far East, while the latter (Opisthorchis viverrini) is home to Thailand, Laos, Viet Nam and Cambodia. Yet another species, Opisthorchis felineus, is more significant for Russia epidemiologically, but it has not been studied well enough yet.

Liver flukes (trematodes) belong to the Opisthorchii-dae family comprising parasites infesting all classes of the vertebrates, mostly mammalians and birds. The main species invading men are the O. viverrini, C. sinensis and O. felineus. Not as much common but just as dangerous are three other species of this family, namely Pseudamphistomum truncatum, Metorchis bilis, and M.xanthosomos.

* Helminths, i.e. worm or wormlike parasites, especially of the intestine, attack the host organism in a variety of ways. Tapeworms and roundworms are helminths (<the Greek helmins, or helminthos, for worm).-Ed.

By different estimates, as many as 40 mln people of Eurasia suffer from trematodosis* caused by these parasites, and yet another 600 to 750 mln run the risk of infestation.

Species of the Opisthorchiidae family exhibit a complex pattern of the life cycle characterized by alternate hosts-two intermediate hosts and one finisher. The first intermediate level takes in Bithynia gastropod snails infecting 23 fish species of the Cyprinidae (carp) family (particularly, the carp, wild carp, bream, roach, etc.). Man and carnivorous animals, the meat and fish eaters, become the final target of the parasites. These are both domestic (dogs, cats, hogs) and wild (foxes, otters, minks, bears, beavers) animals. Man is infested through fish-sliced raw fish, and poorly salted or jerked fish. Then the disease will pardon no one.

Studying Opisthorchiidae, their ecology and biology, under natural and experimental conditions we learn that they are spreading over ever larger areas, and infecting an ever greater number of organisms.

O. felineus and O. viverrini cause opisthorchiasis, or helminthic infestation, affecting mostly the liver, the

* Trematodosis (pl. trematodoses), disease of animals and man caused by infestation with flat parasitic worms, the trematodes (Trematoda), or flukes.-Ed.

gall bladder and bile ducts. In man this disease exhibits no specific clinical symptoms at first but develops fast into a chronic stage characterized by frequent acute conditions; furthermore, it may cause malignancies of the liver and the pancreas (cholangiocarcinomas). Patients infested with such worms run a tenfold risk of hepatic and pancreatic cancers. Liver flukes of the C. sinensis species are responsible for clonorchiosis (clonorchiasis), an illness very close to opisthorchiasis in symptoms, with the bile ducts affected for the most part.

O. felineus, C. sinensis and O. viverrini, in the absence of treatment, may persist for decades, while their population may grow to dozens of thousands because of regular reinvasions.

The incubation period of opisthorchiasis takes between two and four weeks. This is a skittish sickness showing no symptoms at initial stages. Symptoms come on only if the number of parasites in the host organism is large enough, sometimes in ten to twenty years after infestation. Therefore it is rather difficult to diagnose the case and start treatment in good time, and large doses of drugs will be needed then. An eminent Soviet scientist, Konstantin Skryabin (1878-1972), was the first to address the helminthic invasion problem in our country. Still back in the 1920s he masterminded factfinding field expeditions, and often took part in them. There were as many as 350 run-up field parties like that in forty years, covering actually the entire territory of the Soviet Union. They accomplished a remarkable lot in identifying the epidemiological situation countrywide, particularly, in spotting problem areas, and in doing regular prophylactic and health-building work. Great attention was attached to hygienic and sanitary education. The evidence thus collected proved to be of colossal significance for understanding the role of helminths and their fauna in causing pathological conditions in man and animals.

Today the natural seats of opisthorchiasis cover a greater part of the former Soviet republics. Yet it is still problematic to diagnose this disease as a natural phenomenon on account of the growing mobility of the population: O. felineus-infested patients are found far beyond the natural hotbeds of this infection.

TERRA INCOGNITA

The Russian territory accounts for two-thirds of the global opisthorchiasis propagation area. According to a case-study report on the situation in the Russian Federation, as many as 40,000 infected people are registered nationwide every year. Opisthtorchiasis, occurring in 74.8 percent helminthic cases, is still a major social problem.

Opisthorchiasis occurs for the most part in the basins of the Ob, Irtysh, Volga, Kama and Dnieper rivers, but not as often in the basins of the Yenisei, Northern Dvina, and Ural.

These regions contain infestation foci or else hold conditions for helminthic infestation: fish as well as domestic and certain wild animal species were found to be infested; biotypes of Bithynia gastropod snails, the intermediate hosts of O. felineus, were identified there.

According to the worm infestation level, the helminth-invaded areas are divided into four categories: territories of the 1 percent level and below are qualified as zones of sporadic invasions; those of 1-10 percent, 10-40 percent and higher up are characterized as hypo-, meso- and hyperendemic zones, respectively (i.e. exhibiting low, intermediate and high levels of infestation).

The world's largest focus of opistchorchiasis in the Ob-Irtysh river basin is rated as hyperendemic. Here credit is due to Konstantin Skryabin and his field party that spotted this superinfestation zone in 1929. The morbidity rate at different points there is 3 to 28 fold as high as it is countrywide. Even a worse-case situation is registered in the lower and middle reaches of the Ob, where infestation is rampant among the rural population and attains to 90-95 percent, in many cases even preschoolers are infected.

Unfortunately, for all the epidemiological significance of opisthorchiasis, O. felineus is still a way-out field poorly studied by molecular biologists and geneticists. In fact we know hardly anything about the molecular and genetic status of yet another Opisthorchiidae species, Metorchis bilis found alongside O. felineus in mixed infections.

Clearly, such helminthic parasites ought to be closely studied by medics and biologists so as to diagnose all the various cases caused by different liver fluke species and adopt proper medication strategies.

Ο. VIVERRINI ΑΝD Ο. FELINEUS: WHICH WORSE?

O. viverrini is among the species closely related to O. felineus. Since we know about the role of O. viverrini in causing oncological diseases of the liver, it is very important to make a comparative study of these two parasites. This work has been done in a joint effort of the Institute of Cytology and Genetics (RAS Siberian Branch) and Khon Kaen University in Thailand.

Golden hamsters (Mesocricetus auratus), the common model object for opisthorchiasis research, were selected for the experiment. The hamsters were broken into two groups: one was infected with O. felineus, and the other, with O. viverrini. Sound, not infected animals were in the control group. In definite time stretches after infestation their liver was examined for the presence of pathological changes. The O. felineus-infested group showed pathological changes of the liver much sooner, the range of such changes was wider and their symptoms were more explicit than in the hamsters infested with O. viverrini. Thus O. felineus is more aggressive than O. viverrini, and does greater damage to the host organism.

Another major line of our research concerns the cumulative (combined) effect on the liver of O. felineus and dimethylnitrosamine (DMN), a nitrosocompound. Man has to deal with such compounds at every step. Nitrosocompounds get in together with food and water, they are contained in tobacco smoke, and are formed in nitrates and nitrites contained in vegetables and fruit we eat, and produced in metabolic processes triggered by some medical drugs. They are detected in long-shelf life products, such as cheese, smoked foods, and canned meat, fish, fruit and vegetables.

A few years before us, scientists of Khon Kaen University in Thailand found that the combined action of O. viverrini and DMN on animals caused malignant cholangiomas. Yet the carcinogenic effect of O. felineus has not been studied well enough thus far.

We at the Cytology and Genetics Institute have made an experiment to test O. felineus for carcinogenesis. We took 200 golden hamsters in four groups: control (I), DMN-treated (II), O. felineus-infested (III) and those subjected to the combined effect of DMN and O. felineus (IV). The experiment went on for 40 weeks. At definite time intervals we made comparative histological studies of the liver of animals in all these groups.

As expected, the liver condition in the control group saw no changes. The hamsters of the second group exhibited symptoms of progressive hepatic pathology halfway through the experiment, namely adipose infiltration and connective tissue excrescence. Parasites were detected in the bile ducts of the third group. The growing symptoms of pathological processes typical of the precancerous phase were likewise observed there. Overall, the picture was similar to one observed in the experiment conducted together with Thai university research scientists: compared with O. viverrini, O. felineus brings fast and dramatic pathologies of the liver. The situation proved really too bad in the fourth group of hamsters since they were acted upon by both DMN and O. felineus. The inflammatory and degenerative processes observed in the hamsters of the two other experimental groups were manifest earlier and more explicitly. The cholangiocellular carcinoma (malignant tumor) was taking form by the tenth week of the experiment. Given analogous experimental conditions, cholangiocarcinomas appeared but in the fourteenth week in the case of O. viverrini infestation.

This experiment was the first to demonstrate the role of O. felineus in the malignant degeneration of the liver. The "Siberian opisthorchis" is cancerogenic and, like O. viverrini, it acts in synergy with DMN. However, O. felineus is virulent far more than O. viverrini-at any rate, with regard to a model object, the golden hamsters.

PROBLEMATIC DIAGNOSIS

The opisthorchiasis diagnostics problem is still a live issue bearing in mind that the initial stages of the malady at low dosages of the pathogen and even in chronic forms may proceed asymptomatically, while the general clinical picture is not specific. Therefore timely diagnostics is difficult, if possible at all.

To diagnose the "Ob disease" clinicians make use of methods applied in gastroenterology, such as endoscopy, roentgenography, ultrasound, computer and magne-toresonance tomography, among other techniques making it possible to spot structural changes in the liver, gall bladder and biliary ducts, and respective disfunctions.

Since such anomalies may be due to other causes, a preliminary diagnosis has to be restated. But if helminthic ova are detected in a sample, the fact of infestation must be obvious. Yet conventional techniques have

substantial drawbacks-they are laborious and may produce wrong results, for a tentative diagnosis may be checked upon only a month after infestation and egg-laying. The diagnosis may be quite wrong and negative if the invasion level is low, or if the biliary ducts are clogged. It is impossible then to identify a particular parasite and prescribe adequate medication.

Immunoenzymatic analysis (IEA) complements the above methods of opisthorchiasis diagnostics, for it allows to detect in blood serum antibodies specific for antigens of the parasites even at the stage prior to egg-laying, and one can judge about the progress of the disease by the titer of antibodies. Owing to the growing scale of immunodiagnostics, as many as 40,000 cases of opisthorchiasis are registered every year nationwide. However, the available test systems for antibodies to opisthorchis antigens are not perfect. For one, these are low-sensitivity and low-specificity techniques, a factor affecting the fidelity of analysis. Wrongly positive results are also possible in blood serum assays of healthy individuals or patients contracting nonparasitic diseases or parasitic diseases of different etiology (origin).

The accuracy of IEA tests may be improved by a cloning of genes encoding species-specific antigen proteins making it possible to obtain such proteins in sufficient amounts. Proceeding accordingly, we may create high-sensitivity diagnosticums with a specificity level close to 100 percent. Research scientists abroad have indicated certain potential immunogens (antigens) C. sinensis, homologous to antigens of other trematode species by using data on C. sinensis genomic sequencing.

The Cytology and Genetics Institute is working to identify adequate O. felineus antigens on the basis of the latest achievements in genomics, proteomics and the transcription science in conjunction with methods of the bioinformation science. Since there are no data on O. felineus sequencing, we have to resort to transcription analysis covering all RNAs of the parasite, and zero in on a search for the most frequently occurring transcriptions. Such transcriptions have been found, and their data management shows that the translated proteins are homologous to the known antigen proteins of other trematodes, O. viverrini in particular. They likewise have specificity characteristics keeping them apart from these homologues, and thus can be regarded as candidate transcriptions for differential specific diagnostics of O. felineus.

There is yet another side to the diagnostics problem. The point is that helminthiases with similar symptomatology, e.g. metorchiosis and clonorchiosis, may attack in the guise of opisthorchiasis. We know also of mixed infestations exhibiting a complicated clinical picture. Yet the agents of such trematode infestations-O. felineus, O. viverrini, M. bilis and C. sinensis-differ in their biological characteristics, which may tell on the effect of medical drugs. Consequently, we have to identify a parasite and its species before countering with an appropriate treatment strategy.

This problem may be resolved by the method of a polymerase chain reaction (PCR) making it possible to spot a DNA fragment strictly specific for the pathogen. This way we can diagnose the fact of infestation and determine the taxonomic rank of the parasite regardless of its stage of growth. This is a high-performance and relatively low-cost method which, given mass screening, makes it promising for controlling the sanitary-epidemiological and ecological situation. Biotechno-logical firms abroad stepped up work in creating effective PCR diagnostic systems as early as the late 1990s. Our country is not involved in such activities as yet.

The Cytology and Genetics Institute, however, is working toward this end. It has developed and tested a system combining just in "one test-tube" a set of analysis materialized in a multiplex PCR. This system makes it possible to diagnose the very fact of infestation and the agents implicated.

Our diagnosticum has proved its efficiency in practice by diagnosing cases of combined invasions of O.felineus and M. bilis in a test group of patients. If adopted in clinical practice our diagnosticum will certainly boost the diagnostics efficiency.

TREATMENT AND PREVENTION

The available set of medicines for trematodes is rather limited. The basic drug thus used is prasiquantel adopted as far back as 1978. This is a sufficiently effective drug and, given correct and timely diagnostics, it allows to control morbidity in endemic areas. But this drug is not devoid of undesirable side-effects. Furthermore, there are more data on the growing immunity of parasites to it. Consequently, the need for new effective drugs is obvious. Such medicines should rob the parasite of mechanisms it uses in drug detoxification and in developing resistance to medication.

This is how such medication works. Every organism-bacteria and mammalians alike-render harmless xeno-biotics (medical preparations are such foreign substances both for the host and for the parasite) in about the same way, via a universal process of biotransformation involving enzymes of the superfamily of P450 cytochromes. Parasitic internal flatworms, unlike their external counterparts, were hitherto thought to be devoid of enzymes like that. Yet, as we at the Cytology and Genetics Institute have found, O. felineus has one P450 enzyme. Since the biotransformation of xenobiot-ics is of decisive significance for the adaptation of an organism, such minimalism is conditioned by the stability of the habitation medium of O. felineus as an obligate parasite* which thus needs no diversity of biotransformation enzymes. The research into the role of this enzyme in the metabolism of the parasite and in the utilization of prasiquantel is carried on. The results will shed light on the mechanisms implicated in the resistance of O. felineus to antihelminthic medication and make it possible to counter with effective strategies.

One way of combating parasitic worms is to mobilize the internal resources of the host organism, in particu-

* Obligate parasites are unable to survive outside the host organism.-Ed.

lar, its immune system responsible for the resistance to infestations and their aftereffects, bad or not. And so we can prevent this sickness by building up immunity through specialized mono- and polyvaccines depending on pathogenic species home to a particular region. Such prevention offers better advantages compared with conventional pharmacology since vaccines (unless sparking allergies) are free of negative side-effects. It should be stressed in particular that antihelminthic vaccines will also scale down the risk of hepatic, biliary and pancreatic malignancies, the cholangiocar-cinomas.

The authors offer special thanks to Alexei Katokhin, Sergei Shekhovtsov and Kira Zadesenets of the Institute of Cytology and Genetics for the pictorial material kindly provided to us