Institute for Parasitology and International Animal Health, Free University Berlin,
Institute for Biology, Humboldt-University of Berlin, Germany
Federal Research Center for Virus Diseases of Animals, Institute for Epidemiology, Wusterhausen, Germany

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

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Описание случаев описторхоза в Берлине и Федеральной Земле Бранденбург, Германия.

РЕЗЮМЕ
Исключая отдельные случаи заболевания изучение описторхоза в Германии слабо проводилось до 1990 года. Первый осмотр на наличие описторхид, проведённый на 155 беспризорных кошках в восточном Бранденбурге с 1993 по 1995, выявил наличие паразитов у 16% кошек. Проверка 1406 лисиц, застреленных в Федеральной Земле Бранденбург в 1996 году, показала, что описторхоз не ограничивался одним ареалом. Из 460 лисиц (32,7%) с заболеванием печени у 91 (6,5%) обнаружены O. felineus и у 390 (27,7%) были найдены Metorchis bilis. Pseudamphistomum truncatum были обнаружены только у 3 лисиц. Наиболее поражённые печени лисиц (n=314) содержали исключительно M. bilis. Сочетанное заражение M. bilis и O. felineus было выявлено у 74 животных. Среди енотовых собак, проживающих на территории Земли Бранденбург (n=74), выявлено поражение печени только у 8%. В 1997 году из 409 обследованных лисиц в Берлине в 59% случаев обнаружен описторхоз. Вновь наиболее часто встречался M. bilis.

ABSTRACT
Except sporadical findings the occurrence of  opisthorchiid flukes in Germany was poorly investigated before 1990. A first survey on opisthorchiid flukes carried out with 155 stray cats in eastern Brandenburg between 1993 and 1995 showed an Opisthorchis felineus prevalence of  16%. The examination of 1406 red foxes (Vulpes vulpes) shot in the Federal State Brandenburg in 1996 gave evidence that opisthorchiidosis was not restricted to a single focus. Out of 460 (=32.7%) positive for flukes livers 91 (= 6.5%) harboured O. felineus and 390 (=27.7%) were positive for Metorchis bilis. Pseudamphistomum truncatum was found only in 3 foxes. Most positive fox livers (n = 314) contained pure M. bilis infections. Mixed infections between M. bilis and O. felineus were found in 74 animals. Racoon dogs (Nyctereutes procyonoides) (n = 74) originating from Brandenburg state showed a liver fluke prevalence of only 8%. In Berlin even 59% out of 409 examined in 1997 red foxes harboured liver flukes. Again, M. bilis was the most frequently found fluke species. Cyprinid fish acting as second intermediate hosts for this trematode family and serving as infection source for subsequent final hosts had high metacercarial prevalence. Id (Leuciscus idus), roach (Rutilus rutilus), silver bream (Blicca bjцrkna) and bleak (Alburnus alburnus) are considered to be of major importance in the region under investigation.

INTRODUCTION
Liver flukes of the family Opisthorchiidae are widely distributed and cause serious medical problems in several regions where cyprenid fish traditionally is consumed raw or not properly heated. Most human cases are known from the former U.S.S.R. and countries of South East Asia (WHO, 1995). While Opisthorchis felineus, O. viverrini and Clonorchis sinensis are of major medical importance the role of other members of this family in relation to human health is poorly investigated. Already SKRJABIN & SULC (1929) mentioned that Pseudamphistomum truncatum may also be found in humans and MACLEAN et al. (1996) reported an outbreak of Metorchis conjunctus in America.
All these flukes have principally the same live cycle with gill snails as first and cyprenid fish as second intermadiate hosts, respectively. Mammals and humans and or birds act as final hosts.

Little was known about opisthorchiidosis in Germany at the beginning of the 1990. Findings of O. felineus, M. bilis and P. truncatum were made in red foxes (SAAR, 1957, SCHЦFFEL, 1991), in otters (SCHUSTER et al. 1988) and racoon dogs (SCHUSTER et al. 1993). Few cases of human opisthorchiidosis have also been reported from Germany so far (SДNGER et al.1991). Systematic investigations had only started after a finding of O. felineus in a cat originating from a village in the eastern part of the Federal State Brandenburg in 1993.        

      
MATERIAL AND METHODS
Investigations were conducted in the whole Brandenburg State and the city of Berlin. Both federal states stretch over a territory of about 30,500 km2. The Brandenburg State having three regional veterinary institutes in Frankfurt/O., Potsdam and Cottbus is divided into 18 administrative districts and borders to Poland in the east, to Mecklenburg-Pommerantia in the north, to Saxonia-Anhalt in the west and to Saxonia in the south. Berlin with its own diagnostic institute is situated in the centre of the Brandenburg State.
The spectrum of final hosts included stray domestic cats (n = 155) and racoon dogs (n=74) originating from the districts Mдrkisch Oderland and Barnim and red foxes shot in all districts of the Federal State Brandenburg (n = 1406) and the city of Berlin (n = 409). Livers were removed at necropsy at regional veterinary diagnostic institutes and stored at - 20°C. They were kept at room temperature overnight one day prior examination. The gall bladder was opened by scissors and flukes were washed out in 50 ml normal saline. When no flukes were present the sediment of the washing fluid was examined for fluke eggs under a microscope (magnification 100x). Lobules of the liver were sliced into 1 cm slices and by taking these slices between two fingers flukes were squeezed out of the bile ducts in a Petri dish. After removal of the remnants the sediment was poured off and flukes were counted and determined. For statistical analyses the software package Quantitative Parasitology 1.0 by ROZSA et al. (2000) was used.

In addition, 1079 specimens of 7 species of fish, namely roach (Rutilus rutilus), bream (Abramis brama), silver bream (Blicca bjoerkna), blue bream (Abramis ballerus), id (Leuciscus idus), bleek (Alburnus alburnus) and rudd (Scardinius erythrophthalmus) caught in waters of Brandenburg State and Berlin were examined for muscle metacercariae. In a first investigation muscle samples were squashed between two glass plates and metacercariae were counted under a trichinelloscope while in further investigations muscle flesh was digested in artificial gastric juice and the sediment was checked for metacercarial cysts under a stereoscopic microscope. For differentiation a key by SCHUSTER et al. 1998 was used. Due to difficulties to separate cysts of O. felineus from M. bilis by morphological features metacercariae of mammalian opisthorchiids where grouped together.  

RESULTS
24 (= 16%) out of 155 cats dissected were positive for liver flukes. All of them harboured O. felineus in numbers between 1 and 1370 specimens. Beside O. felineus 4 cats had single specimens of M. bilis.
6 (= 8%) out of 74 racoon dogs examined were positive only for M. bilis.
The large number of examined foxes originating from all administrative districts allowed a much more differentiated evaluation. For the reason of clearness data were summarised and the area under investigation was divided into the eastern, western and southern parts of the Brandenburg State and the city of Berlin. As seen from Tab. 1 the  foxes in the 4 subareas showed significant differences in the general liver fluke prevalence. The lowest prevalence was found in the southern districts of Brandenburg State and M. bilis was the only species identified. The highest general fluke prevalence was found in foxes originating from Berlin. Compared to the other subareas foxes in Berlin also showed significant higher prevalence of O. felineus and M. bilis while in the case of P. truncatum no significant differences were found.
If we consider only those foxes with findings of adult flukes a similar species composition can be seen in animals originating from the eastern and western Brandenburg State and those from Berlin (Tab. 2 – 4). More than 90% of positive animals were infected with M. bilis. The percentage of O. felineus infections varied between 22 and 29% mostly being combined with M. bilis.

Regarding mean fluke intensities significant differences were found by comparing O. felineus in positive foxes from Berlin with those originating from the eastern districts of Brandenburg State (Tab.5) although the prevalence of this fluke species was higher in Berlin. Contrary to O. felineus, mean intensity of M. bilis rose significantly from 10 in the eastern part to 32 in the western part of Brandenburg State to 62 in Berlin. The significance also could be proved by bootstrap tests for mean intensities and Mood’s median tests (p<0.05).

By grouping the number of flukes into intensity classes 80 to 85% of the foxes showed low burdens in the case of O. felineus while in the case of M. bilis higher intensities were associated with growing prevalence (fig 1 and 2). Thus, in the eastern districts of the Brandenburg State only 12% of foxes harboured more than 20 flukes while in the western districts more than 20 flukes were isolated from 30% of the positive foxes. In Berlin, even 44% of positive foxes were grouped in the highest intensity class.
Metacercariae of mammalian opisthorchiids were found in all fish species examined. The highest prevalence was found in roaches (up to 88%), in silver breams (up to 81%), in ids (up to 94%) and in bleeks (up to 99%). Ids harboured the highest burdens of muscle metacercariae in their flesh. The maximum density established was 69 per g. While cysts of the O. felineus/M. bilis-complex were found in all infected fish specimens P. truncatum metacercariae were established in low densities in a much lower prevalence.

DISCUSSION
There are two simple ways to establish the presence of opisthorchiid flukes in a given region: examination of susceptible cyprenid fish for muscle metacercariae or examination of susceptible final hosts. Going the first way species determination in the case of metacercariae of O. felineus and M. bilis is possible only by infection of experimental hosts, while those of P. truncatum can be determined easily by morphometric parameters. In final hosts species differentiation is possible only at necropsy. However, a newly developed antibody detecting ELISA gave promising results to differentiate pure O. felineus infections from M. bilis infections (DELL et al. 1999).

Although domestic cats were recommended by CHAVKIN (1989) as bioindicators for the presence of O. felineus our findings showed that the red fox was a much more suitable host for larger epidemiological investigations. Large numbers of foxes have been shot and sent to diagnostic institutes in connection with rabies control programmes. All foxes got their parasitic infections by a natural way while cats acquire liver flukes mainly when they are fed with fish by their owners. This may falsify the whole picture especially in cases when positive cats originated from places where water bodies where absent. In favour of using red foxes as indicator hosts is also the fact that foxes have a relatively small home range and places of bagging in most cases are identically with the place where the liver fluke infection had taken place. Although racoon dogs more often occur in river meadows as typical biotopes for opisthorchiids according to our findings they seemed to be not as suitable as foxes for this purpose. On the one hand prevalence of racoon dogs with opisthorchiids was much lower on the other hand due to their migrating behaviour and larger home ranches it is much more difficult to get to know where the infection actually has taken place. However, racoon dogs can be considered to act as distributors of opisthorchiid flukes.

How to explain differences in the prevalence of liver flukes in foxes originating from different regions? Obviously several reasons have to be considered. One main reason is the presence and the access for foxes to surface waters in a given area. This is directly correlated with the availability of cyprenid fish. The percentage of  water surface in the eastern, western and southern parts of Brandenburg State and the city of Berlin is 4.8, 3.2, 2.8 and 5.3% respectively. This may explain the lowest opisthorchiid prevalence in the southern Brandenburg and the highest in Berlin.

Although the red fox is a food generalist it mainly feeds on voles. In areas where voles are not available in larger amounts foxes have to search for alternative food sources including fish. This probably is the case in the outskirts of Berlin where fish catching foxes were observed. In the inner districts of Berlin due to the high shores of the canals and the river Spree left overs of fishermen are the only source of liver fluke infection.

Regarding the first intermediate hosts M. bilis and P. truncatum use Bithynia tentaculata. O. felineus requires B. leachi. While Bithynia tentaculata is found in most of the natural waters and canals in Brandenburg and Berlin B. leachi belongs to the rarely occurring snail species in the region and large populations are only found north of Brandenburg State. This fact may explain why M. bilis in foxes occurred in higher prevalence and intensities in comparison to O. felineus. On the other hand the spectrum of final hosts of M. bilis is not only restricted to mammals and man. The fact that it also can infect fish eating birds increases the intensity of the life cycle of this parasite. No explanation can be given for the rare findings of P. truncatum in final hosts and as metacercariae in cyprenid fish.

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