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Echinococcus granulosus in Finland
Veterinary Parasitology 111 (2003) 175–192
Echinococcus granulosus in Finland Varpu Hirvelä-Koski a,∗ , Voitto Haukisalmi b , Seija-Sisko Kilpelä c , Minna Nylund a , Perttu Koski a a
b
National Veterinary and Food Research Institute (EELA), Oulu Regional Unit, P.O. Box 517, FIN-90101 Oulu, Finland Finnish Forest Research Institute, Vantaa Research Center, P.O. Box 18, FIN-01301 Vantaa, Finland c Finnish Game and Fisheries Research Institute, Taivalkoski Game and Fisheries Research, Ohtaojantie 19, FIN-93400 Taivalkoski, Finland Received 20 June 2002; received in revised form 15 October 2002; accepted 16 October 2002
Abstract Echinococcus granulosus is shown to occur in eastern Finland in a sylvatic cycle involving wolves (Canis lupus) as the definitive host and reindeer (Rangifer tarandus tarandus) and probably also elk (Alces alces) and the wild forest reindeer (Rangifer tarandus fennicus) as intermediate hosts. Even though the prevalence of E. granulosus in reindeer has increased in recent years, it is still very low (<0.013%). The results suggest, however, that the reindeer is a good sentinel animal to show the contamination of soil with Echinococcus eggs. This is the first report of wolves acting as a definitive host for E. granulosus in northern Europe. The parasite seems to be quite common in the Finnish wolf population, with a prevalence of approximately 30% in both intestinal and faecal samples. The present results and previous ones indicate that E. granulosus infection has not spread to dogs in the reindeer herding area. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Echinococcus granulosus; Cestoda; Reindeer; Wolf; Dog; Finland
1. Introduction Echinococcus granulosus has occurred earlier as an endemic infection in reindeer herding area in the northern parts of Fennoscandia, where the life-cycle involved the dog as the definitive host and the reindeer (Rangifer tarandus tarandus) as the intermediate host (Skjenneberg, 1959; Pöysti and Pöysti, 1969; Roneus, 1974; Kummeneje, 1982). The prevalence in reindeer varied between regions, the highest values being 10–14% (Skjenneberg, ∗ Corresponding author. Tel.: +358-8-5622-633; fax: +358-8-5544-977. E-mail address: [email protected] (V. Hirvelä-Koski).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 3 8 1 - 3
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1959; Söderhjelm, 1946). There are no reports on the rate of infection of dogs in this area, however, E. granulosus infections have occasionally been found in humans, mostly in Lapps living in close contact with their dogs, which were used for shepherding the reindeer (Söderhjelm, 1946; Cederberg, 1946; Brüning, 1948; Arnesen, 1953; Myrseth, 1956; Rein, 1957; Lindholm and Lantto, 1968; Huldt et al., 1973). However, in Finland no domestic incidences of E. granulosus infection in humans have been encountered for several decades (Maijala et al., 2002). Circumstances in northern Finland have changed since the 1960s, due to obligatory inspection of all reindeer meat since 1975, better hygiene in handling the slaughter offal and a higher standard of living among the reindeer owners. The use of shepherd dogs has also diminished since snowmobiles were adopted for rounding up the reindeer herds from the 1960s onwards. However, since 1992 E. granulosus cysts have again been regularly found in reindeer carcasses in connection with meat inspections. It is not known whether this is due to a true increase in its prevalence or to increased efficiency of meat inspection. The first abattoir for reindeer slaughtering was built in 1984, but it was not until 2001 that all reindeer to be slaughtered were transported to abattoirs and inspected there. Hydatid cysts have been found both at abattoirs and at slaughtering places in the field. The occurrence of echinococcosis in livestock in Finland is monitored in connection with meat inspection. Hydatid cysts have occasionally been found in horses and in one cow (National Veterinary and Food Research Institute, Finland, unpublished data), but never in sheep, goats or pigs. Echinococcus multilocularis has never been found in Finland (Maijala et al., 2001). We describe here the occurrence of hydatid cysts in reindeer in Finland and report on the results of examinations of potential definitive hosts for E. granulosus, i.e. dogs and wolves. 2. Materials and methods 2.1. Areas studied Areas 1, 2 and 3 are situated along the eastern border of Finland (Fig. 1). Other parts of the country are designated as area 4. Areas 1 and 2 are in the reindeer herding area. Area 1 is located between 27◦ 30 and 29◦ 30 E and 70◦ and 68◦ 30 N, area 7586 km2 consisting half of the municipality of Inari, population density being 0.5 inhabitants/km2 . Area 2 consists of municipalities Salla, Suomussalmi, Hyrynsalmi and Kuusamo, located between 28◦ and 30◦ 30 E and between 67◦ 45 and 64◦ 30 N, area 17 445 km2 and the population density ranging from 0.9 to 3.5 inhabitants/km2 . Area 3 is located between 26◦ 30 and 31◦ 30 E and between 64◦ 30 and 60◦ 20 N, consisting of 28 municipalities, the area being 26 830 km2 and the population density 2.0–35.2 inhabitants/km2 in rural parts and up to 522.3 in the cities. The areas 1–3 all represent northern boreal forest. The forests in area 1 are dominated by pine (Pinus sylvestris), and in areas 2 and 3 by spruce (Picea abies) and pine. Birches (Betula pendula and B. pubescens) are the most common deciduous trees in all areas. The average temperature of July is ca. +10 ◦ C in area 1, between +10 and 15 ◦ C in area 2
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Fig. 1. Geographical distribution and numbers of wolf samples in 1999–2001. The first column: number of samples; the second column: number of positive samples. Symbols: ( ) dried faecal samples; ( ) frozen faecal samples; ( ) intestinal samples.
and ca. +15 ◦ C in area 3. The yearly precipitation varies between 500 and 700 mm, being lowest in area 1. In areas 1, 2 and 3 the length of the growing season is 100, 100–150 and 150–220 days and the length of snow cover 200–250, 150–200 and 100–150 days per year, respectively. 2.2. Examination of farm livestock and reindeer In Finnish slaughterhouses, the carcasses of semi-domesticated reindeer (R. tarandus tarandus) and farm livestock, including cattle, sheep, goat, horses and pigs, are routinely examined for Echinococcus metacestodes during meat inspection. When hydatid cysts are suspected, they are sent to the National Veterinary and Food Research Institute to be examined for echinococcosis in terms of gross pathology, microscopy and histology. These examinations provide descriptions of the appearance of the cysts and their elasticity, size, number and location and exclude the possibility of other parasitic infections or cysts of non-parasitic origin. The cyst contents and scrapings from the cyst wall are examined for Echinococcus protoscoleces of characteristic morphology by microscopy. For sterile and degenerated cysts, histological examination is performed using haematoxylin-eosin staining to show the typical structures of the cyst wall, with an outer acellular laminar
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hyaline layer and a thin syncytial germinal layer. The cyst is surrounded by a fibrous capsule and infiltrate of inflammatory cells, including giant cells and eosinophils. In the case of degenerated cysts, the diagnosis is confirmed by identification of the outer laminar layer. All information concerning the infected reindeer during 1979–2001 were obtained from the diagnostic data of National Veterinary and Food Research Institute. The numbers of reindeer slaughtered annually in 1979–2000 were obtained from the official statistics of the Association of Reindeer Owners. 2.3. Wolves 2.3.1. Dried faecal samples A total of 110 faecal samples were collected from areas 3 (n = 109) and 4 (n = 1) between March and October 1999 (Fig. 1) and kept on an open petri dish at room temperature until they were dry and their weight was stable. After examination for taeniid eggs by the flotation method, the samples were stored in a frozen state at –20 ◦ C. Thirty of them were examined for Echinococcus coproantigen by ELISA (Bommeli Diagnostics), the average time elapsing between collection and the flotation test being 86 days (range 47–268 days). 2.3.2. Frozen faecal samples A total of 332 faecal samples were collected from the field in the years 1999–2001. Nearly all of them (n = 330) were from area 3 and the rest (n = 2) from area 2. Most of the samples represented one animal, but there were also some pooled samples. In order to study the life habits of wolves, the alpha pair and some juvenile wolves in several packs had been radio-collared and followed by radiotelemetry (Kojola, 2002, personal communication). In 2001, for instance, there were 17 radio-collared wolves and five family groups under observation in this way. On the basis of this information the faecal samples could often be identified to a certain group of wolves and samples could be collected especially around the dens, from daily resting places after the wolves had left and from around prey animals 2–3 days after the kill. The age of the sample at the time of collection was estimated by the field worker. Before arriving at the laboratory most of the samples had been frozen for storage purposes, however, and the conditions of preservation in the field were not documented. In the laboratory the samples were kept frozen at −20 ◦ C until tested for Echinococcus coproantigen by ELISA (Bommeli Diagnostics). The average time elapsing between collection and arrival at the laboratory was 64 days (range 0–365 days), and that between arrival and ELISA testing 326 days (range 36–539 days). 2.3.3. Intestinal samples Intestinal samples were obtained from 23 wolves in 1999–2001. There were 12 males and 11 females. The wolves originated from areas 1, 2 and 3, near the eastern border of Finland (Fig. 1). Twenty of the wolves had been killed during hunting, two had been found dead in the field and one was shot after a traffic accident. In 11 cases the small intestine, large intestine and rectum were immersed in 5% formaline, in 11 cases they were frozen and in one case the whole carcass was frozen at −20 ◦ C (Table 3). The formaline-fixed samples were examined for Echinococcus worms by the sedimentation method, while the
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frozen samples were first examined for Echinococcus coproantigens with ELISA (Bommeli Diagnostics ), and those giving positive or ambiguous ELISA values were tested further by the sedimentation method. Seven of the frozen samples were also examined for taeniid eggs using the flotation test. 2.4. Dogs All the dog samples were collected from area 2, in the eastern part of the reindeer herding area (Fig. 1). Faecal samples were obtained from 352 dogs in co-operation with local veterinarians in the years 1999 and 2001. The sampling period extended from February to May in both years. Most of the samples (68%) were obtained from dogs used for hunting and five from ones that had been used as shepherd dogs in reindeer herding. Antiparasitic drugs had been given to 61% of the dogs during the year before sampling. About 20% of the dogs had been treated with drugs containing praziquantel. The fresh samples taken in 1999 (n = 169) were kept cool and sent to the laboratory once a week. After examination using the flotation test, they were kept frozen at −20 ◦ C until tested for Echinococcus coproantigen with ELISA (Genzyme Virotech GmbH). Samples giving positive or ambiguous results were re-tested with Bommeli Diagnostics ELISA. In 2001, the fresh samples (n = 183) were kept frozen at −20 ◦ C until examined for Echinococcus coproantigen with ELISA (Bommeli Diagnostics) and for taeniid eggs by the flotation method. Repeat samples were tested in the case of seven dogs. 2.5. Flotation method The faecal sample was homogenised by manual pressing in a plastic bag, 3.0 g was weighed out, diluted to 42 ml with tepid tap water, mixed thoroughly and strained through a tea sieve. After centrifugation of 12.5 ml of the filtrate (660 × g, 3 min), the supernatant was removed until there was approximately a 2 mm layer of fluid above the sediment. Concentration solution (NaCl 393 g, saccharose 50 g, water 1000 ml, specific gravity 1.21) was added to a total volume of 2.5 ml and mixed thoroughly. A Mc Master counting chamber was filled with the solution and the number of eggs was counted under low magnification. 2.6. Sedimentation method The sedimentation method was performed as described by Eckert et al. (1984). As a safety precaution, the frozen samples were moved to −70 ◦ C for 7 days before examination. After thawing, the small intestine was divided into 4–6 sections, each of which was split longitudinally and immersed in physiological saline at 37 ◦ C. After 30 min, the intestinal wall was scraped and the scrapings were added to the solution. After sieving (mesh size 1.5 mm), the solution was poured into a high vessel and left to settle for 30 min. The supernatant was removed with a pipette. The sedimentation was repeated several times to remove the flocculant material. A small amount of the sediment at a time was poured into a black tray and examined for adult Echinococcus worms or their fragments under a stereo microscope. The number of worms was determined by the number of Echinococcus scoleces found.
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For specific identification of Echinococcus, several entire or fragmented strobilae from two wolves were stained with Semichon’s acetic carmine, cleared in eugenol and mounted in Canada balsam. 2.7. ELISA for the detection of Echinococcus-specific coproantigens Two commercial kits were used for the detection of Echinococcus-specific coproantigens, Echinococcus ELISA (Genzyme Virotech GmbH, Rüsselsheim, Germany) and Echinotest (Bommeli Diagnostics, Liebefeld-Bern, Switzerland). The tests were performed according to the manufacturers’ instructions. All faecal samples were frozen for 7 days at −70 ◦ C before examination. In the case of frozen intestines, the sample was taken either from the rectum or from the posteriormost part of the large intestine. All samples were tested in duplicate. In Genzyme Virotech ELISA, values <9.0 were interpreted as negative, 9.0–11.0 as ambiguous and >11.0 as positive, while in Bommeli Diagnostics ELISA values below 30 were regarded as negative, 30–40 as ambiguous and values exceeding 40 as positive. If the ELISA result was positive or ambiguous, the test was repeated once or twice, the values for the different test runs being averaged to obtain the final result. 2.8. Statistics The underlying assumptions for the parametric tests were often invalid because the data were asymmetrical or not normally distributed, and non-parametric tests were therefore used. The Mann–Whitney U-test was performed to compare the time interval from collection to freezing between the wolf samples with positive and negative ELISA values. Spearman’s rank correlation was used to test for an association between the estimated ages of the wolf samples and their corresponding ELISA values. The number of infected reindeer among the adult animals slaughtered in 1992–1995 in area 2 was compared with the situation in 1996–1999 by means of the χ2 -test, which was also used to test the differences in the proportion of infected wolves in five family groups. Statistical analyses were carried out using the analytical software package Statistix for Windows (Analytical Software, 2000).
3. Results 3.1. Prevalence of E. granulosus in farm animals and reindeer E. granulosus cysts have been found in Finland during the years 1979–2001 in five horses and one cow (unpublished data from National Veterinary and Food Research Institute, Finland). The positive horses found in 1998 and 1999 were from area 4 and the cow, found in 1986, from area 3 (Fig. 1). The geographical origins of the positive horses found in 1979, 1992 and 1994 are unknown. Echinococcosis was reported in one reindeer during the period of 13 years from 1979 to 1991 (Table 1), but cases have been diagnosed annually since 1992, and a total of 59 reindeer had been shown to have been infected with hydatid cysts by 2001. The proportion of infected animals in the whole population of slaughtered reindeer ranged between 0.008
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Table 1 Number and proportion of reindeer infected with Echinococcus granulosus in all slaughtered reindeer and among adult animals (>1 year of age) in Finland in 1979–2001 Year(s)
Number of reindeer infected
Infections per 1000 reindeer
Infections per 1000 adults
Number of slaughtered adults
1979–1986 1987 1988–1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001
0 1 0 1 1 1 4 1 10 10 12 6 13
– – – 0.008 0.008 0.008 0.033 0.012 0.111 0.104 0.129 0.069 –
– – – 0.023 0.026 0.030 0.118 0.037 0.391 0.382 0.453 0.233 –
– – – 42 751 37 892 33 397 33 938 27 369 25 586 26 156 26 479 25 781 –
Statistics on the numbers of reindeer slaughtered are not yet available for 2001.
and 0.129 per 1000 animals yearly, and that among adult animals (>1 year) between 0.023 and 0.453 per 1000 (Table 1). In both groups, there seems to have been more than 10-fold increase in the proportion between 1994 and 1997. The positive animals originated mostly from eastern border, close to Russia, except for one case from the western part of the reindeer herding area in the year 1999 (Fig. 2). During the period 1992–1995 there were 7 reindeer with hydatid cysts out of a total of 17 995 adult animals slaughtered in area 2, the corresponding figures in 1996–1999 being 32 out of 12 434. The χ2 -test showed a statistically significant increase in the number of infected adult animals found in connection with meat inspection between the two periods (P < 0.001). There were at least two distinct clusters in the occurrence of hydatid cysts in area 2, the proportion of positive cases in the more northerly one in 1995–1999 being 2.1 per 1000 reindeer slaughtered or 7.5 per 1000 adult reindeer slaughtered. Information on age and sex was obtained with respect to 27 infected reindeer, including 23 adult females, 2 adult males and 2 juveniles (less than 1 year old). The exact age was known in 12 cases, 25% being 3–7 years old and 75% 9–11 years old. The site of the cysts was most often in the lung (89.8%), but the liver and occasionally the spleen were also involved (liver 5.1%, liver and lung 3.4%, spleen and lung 1.7%). The number of cysts detected per animal varied between one and seven and the diameter between 0.5 and 10 cm. Most of the animals (52.5%) harboured one cyst, 23.7% two cysts, 15.3% three cysts, 6.8% four cysts and 1.7% seven cysts. Degenerated or calcified cysts were detected in six reindeer (10%). In 45 reindeer (76%), the cysts were fertile and direct microscopy of the contents revealed E. granulosus protoscoleces. The diameter of the fertile cysts was 3–8 cm in 31 cases (69%) and more than 8 cm in 13 cases (29%). Only one of the fertile cysts (2%) was less than 3 cm in diameter. In 14 cases, no protoscoleces were found and the diagnosis was based on histological examination. The sterile cysts were smaller, their diameter being under 3 cm in 77% of the cases, although the largest again measured 10 cm.
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Fig. 2. Geographical distribution of echinococcosis detected in Finnish reindeer during meat inspection in 1992–2001. Each dot marks one infected reindeer (n = 58).
3.2. Wolves 3.2.1. Dried faecal samples All the dried faecal samples collected in 1999 were negative for taeniid eggs in the flotation test, but eight contained Toxocara sp. eggs 20–1200 EPG. Three of the 30 samples tested with ELISA gave positive results, with values of 128, 75 and 73. Flotation testing of the positive samples did not reveal any signs of parasitic infections. The time between collection and freezing had been 73–78 days, and that between freezing and testing 494–577 days. 3.2.2. Frozen faecal samples The distribution of ELISA values for the frozen faecal samples is shown in Fig. 3. A total of 96 of the samples (29%) were positive in the ELISA test, with values ranging from 40 to
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Fig. 3. Distribution of ELISA values for faecal samples from wolves (n = 332) and dogs (n = 183) tested for Echinococcus coproantigen by Bommeli Diagnostics ELISA.
242. There were 13 samples (4%) giving ambiguous values between 30 and 40, while most of the negative samples (86%) gave values below 21. ELISA results for six family groups (packs) are presented in Table 2. All the samples for packs 3 and 6 were negative in ELISA. The number of samples examined in the negative packs was nevertheless too small to allow any definitive conclusion to be reached regarding the absence of E. granulosus infection in these groups, but the χ2 -test result did indicate a slight difference between the groups (0.05 < P < 0.1). The temporal distribution of the samples from groups 1, 2 and 5 is presented in Fig. 4. No seasonal variation could be observed in the prevalence of E. granulosus. One wolf in group 1, two in group 2 and one in group 3 had been killed during 2001 (Table 3, numbers 9, 13, 14 and 20, respectively). In two of them (in groups 1 and 2) E. granulosus was detected in the intestine by sedimentation method, the approximate numbers of worms being 110 and 1670, respectively. All the samples giving ELISA values >30 were re-tested. Of the 95 samples positive in the first test run, 92 (97%) remained positive after the second run, while three samples with low ELISA values (43, 44 and 47) gave negative or ambiguous values in the second run (29, 19 and 30, respectively). Seventeen samples gave ambiguous ELISA values in the first test run, six of which became negative in the second run, six still gave ambiguous values and five became positive. The age of the faecal mass at the time of collection was assessed by the collector. Most of the samples were estimated to be less than 8 days old, the variation being from <1 day to 1–2 months. There was no significant correlation between the ELISA value and the age among these 0–7 days old samples (Spearman’s rank correlation coefficient −0.023, n = 225, P > 0.05). The oldest positive samples were estimated to be 10–14 days and 1–2 months old, with ELISA values of 185 and 76, respectively. The median time elapsing between collection and freezing in the laboratory was 53 days (range 4–365 days) for the samples with a positive ELISA result and 49.5 days (range 0–325 days) for the negative samples.
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Pack number
Collecting period
1 2 3 4 5 6
January 2000 to May 2001 September 1999 to May 2001 May 2000 to December 2000 January 2001 to July 2001 May 2001 to September 2001 August 2000 to September 2001
Total
Number of samples
Number of positive samples
Percentage of positive samples
71 98 6 2 42 6
27 36 0 2 11 0
38 37 0 100 26 0
225
76
34
Number of ambiguous results
Percentage of ambiguous results
Range of negative ELISA values
Range of positive ELISA values
7 2 0 0 1 0
10 2 0 0 2 0
0–27 0–29 2–23 – 0–21 0–29
44–213 43–242 – 92–112 43–129 –
10
4
–
–
All samples giving positive or ambiguous test results were re-tested once or twice and the values for the different test runs were averaged to obtain the final result.
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Table 2 ELISA (Bommeli Diagnostics) results for six family groups (packs) of wolves
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Fig. 4. Seasonal variations in the number of faecal samples and ELISA results for three family groups (packs) of wolves in 2000–2001.
This difference was not statistically significant in the Mann–Whitney U-test (U = 12 808, n = 323, P > 0.05). 3.2.3. Intestinal samples The results of the examinations of the intestinal samples are shown in Table 3 and Fig. 1. Echinococcus worms were found by the sedimentation method in six wolves, while eight
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Table 3 Results of intestinal samples from wolves Date of death
Area
Preservation of the intestine
Sedimentation method
Approximate number of worms
Identification of E. granulosus
ELISA result (%)
Flotation method
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
20.11.1999 10.2.2000 27.10.2000 27.10.2000 30.10.2000 31.10.2000 16.1.2001 19.1.2001 19.1.2001 23.1.2001 25.1.2001 25.1.2001 25.1.2001 25.1.2001 26.1.2001 28.2.2001 2.3.2001 3.3.2001 5.3.2001 9.3.2001 15.3.2001 31.5.2001 2.6.2001
2 3 2 2 3 1 3 3 3 3 2 2 2 2 2 2 2 2 3 3 3 3 3
Formaline-fixed Formaline-fixed Formaline-fixed Formaline-fixed Formaline-fixed Formaline-fixed Formaline-fixed Formaline-fixed Formaline-fixed Frozen Formaline-fixed Frozen Frozen Frozen Formaline-fixed Frozen Frozen Frozen Frozen Frozen Frozen Frozen Frozen
Positive Positive Negative Negative Negative Negative Negative Positive Positive ND Negative ND Negative Positive Negative ND ND ND ND ND ND ND Positive
5 364 0 0 0 0 0 5 110 – 0 – 0 1670 0 – – – – – – – 718
× × – – – – – – – – – – – – – – – – – – – – –
ND ND ND ND ND ND ND ND ND 12 ND 6 76 119 ND 16 6 8 0 11 0 14 115
ND ND ND ND ND ND ND ND ND Negative ND ND ND Negative ND Negative Negative ND Negative ND Negative ND Negative
Bommeli Diagnostics ELISA was used for the detection of Echinococcus coproantigen.
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Number
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were negative, the positive wolves having been killed in areas 2 and 3. Because of the abundance of flocculent material in the sediment in spite of several washings, only a small amount of the sediment could be examined under the microscope at a time, which meant that only 20% of the sediment was examined in case of the negative samples. The proportion of the sediment examined in the case of the positive samples varied between 10 and 50%. The number of worms was determined by relating the number counted to the volume of sediment examined. The estimated number of worms per infected wolf varied between 5 and 1670. Three of the frozen samples were positive in ELISA, giving values of 76, 115 and 119, but in one of these, with an ELISA value of 76, no worms could be detected by the sedimentation method even though the whole sediment in this sample was examined. Nine samples were negative in the ELISA test, with values ranging between 0 and 16, and there were seven samples examined by the flotation method in which no taeniid eggs were found. The stained and mounted adult specimens of Echinococcus from wolves usually lacked rostellar hooks, but their other morphological features, particularly the position of the genital pore in mature segments (at the mid-level), and the number (ca. 50) and distribution of testes (anterior and posterior to the level of the genital pore) showed unambiguously that these specimens represented E. granulosus, rather than E. multilocularis (see Rausch, 1956 and Thompson, 1995 for the identification of Echinococcus species). 3.3. Dogs All the faecal samples from dogs were negative for taeniid eggs in the flotation test in both years. The first sampling in 1999 (n = 169) included six positive samples in ELISA and two with an ambiguous result, but they all became negative in the second test run. The ELISA values varied between 9.9 and 28.6 (Genzyme Virotech) in the first run and between 0 and 25 in the second (Bommeli Diagnostics). Although the ELISA values of the negative samples varied from 0 to 8.8, most of the negative samples (152/161) gave values lower than five with the Genzyme Virotech test. Ten samples out of the 183 taken in 2001 (6%) were positive and 7 (4%) gave ambiguous values based on the averaged results of the two test runs with Bommeli Diagnostics ELISA. The distribution of ELISA values is shown in Fig. 3. The negative values varied between 0 and 29 and the positive ones between 43 and 62. Of the 12 samples giving ambiguous values in the first test run, 6 became negative in the second run, 3 remained ambiguous and 3 became positive. Of the seven positive samples, six remained positive and one became ambiguous in the second run. No taeniid eggs were found in any of the samples by the flotation method, nor did this test reveal any other parasitic infections in the dogs gaining false positive or ambiguous values in ELISA. Repeated samples obtained from seven dogs with positive ELISA values varying between 35 and 62 in the first sampling were all negative, with ELISA values ranging from 4 to 26. The time elapsing between the two samplings was 25–43 days. All except one of the dogs initially giving a positive value were used for hunting, and only one of these had been given antiparasitic drugs effective against Echinococcus within a year of sampling.
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4. Discussion This is the first report of wolves acting as a definitive host for E. granulosus in northern Europe. The sylvatic life-cycle in Finland involves wolves and cervids, i.e. reindeer, and most probably also the elk (Alces alces) and the wild forest reindeer (Rangifer tarandus fennicus), which are the main prey species for wolves in eastern Finland. The parasite seems to be quite common in the Finnish wolf population, the results for both the intestinal and faecal samples indicating a prevalence of approximately 30%. This figure is comparable to the infection rates of wolves reported from North America and the Russian far east, ranging from 14 to 72% (Rausch, 1995). There was some clustering among the family groups, probably as a natural consequence of the custom of wolves to prey as a pack (Table 2). There was no clear seasonal variation in the occurrence of E. granulosus in wolves (Fig. 4). The minimum population of wolves in Finland was 98 animals in 1999, 130 in 2000 and 120 in 2001 (Kojola, 2000, 2001). The occurrence of E. granulosus in livestock throughout Finland has been monitored in accordance with the meat inspection instructions since the 1970s. Hydatid cysts have been occasionally found in horses and in one cow, but never in sheep or pigs, even though there are farms rearing both sheep and cattle even in areas 1, 2 and 3. In the case of the positive cow found in area 3 in 1986, wolves were suspected as the possible origin of the infection. The horse infections seem to have no epidemiological connection with infections by the Scandinavian cervid strain. The present figures describing the infection rate in reindeer are very low compared with those reported for other regions where the northern biotype of E. granulosus occurs in the sylvatic cycle. In parts of Canada, where the wolf is the chief natural primary host, 9–15.5% of the reindeer harboured hydatid cysts (Cameron, 1960), while Thomas (1996) reported that 2% of the caribou in the 2–5 years class and 8% of the older individuals were infected. Rausch (1995) reported prevalences of 3 and 5% in wild reindeer in arctic Alaska. In elk or moose (A. alces), prevalences of the northern biotype ranging from 24 to 59% have been reported in North America, and a figure as high as 71% in the far east of Russia (Rausch, 1995). In Fennoscandia, the prevalence in semi-domesticated reindeer has been 1.2–1.6% (Pöysti and Pöysti, 1969; Roneus, 1974; Kummeneje, 1982), higher figures can be found in older reports (9.6% in Norway, Skjenneberg, 1959). There are no permanent family groups of wolves in the reindeer herding area in Finland, and single wolves moving about in the area are killed rapidly in order to avoid reindeer losses. The absence of a permanent high-density population of the definitive host probably explains the low infection prevalences in reindeer in Finland. The reindeer pasture most of the year free in the forests and marshlands feeding on grass, sedge, mushrooms, lichen and other plants growing low near the ground. Because of its feeding habits, semi-domesticated reindeer seems to be a good sentinel animal to show contamination of earth and plants with eggs of the northern biotype of E. granulosus. In addition, hydatidosis in reindeer can easily be detected and diagnosed via the meat inspection process performed by the local veterinarians. Dogs can also serve as a definitive host for the northern biotype of E. granulosus (Cameron, 1960; Sweatman and Williams, 1963; Wilson et al., 1968; Rausch, 1995), and they are considered the main source of human infections in endemic areas where there is a
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sylvatic life-cycle of E. granulosus with wolves as the primary definitive hosts (Cameron, 1960; Wilson et al., 1968). Dogs have been regarded as the source in all reports on human infections in the northern parts of Fennoscandia (Söderhjelm, 1946; Cederberg, 1946; Brüning, 1948; Arnesen, 1953; Myrseth, 1956; Rein, 1957; Lindholm and Lantto, 1968; Huldt et al., 1973). According to the present results, E. granulosus has not spread to dogs. Dog samples from area 3 (Fig. 1) should also be examined, however, to confirm the absence of E. granulosus in dogs. Coproantigen detection by enzyme-linked immunosorbent assay (ELISA) has been deemed a practical method for screening E. granulosus in the definitive host because of its high sensitivity and specificity and high probability of correlation with current infection (Eckert et al., 2001a). ELISA detects infected animals within the pre-patent period and converts to negative in a few days after praziquantel treatment (Deplazes et al., 1992). Genzyme Virotech place the sensitivity and specificity of their ELISA test at 76 and 95%, respectively, while Bommeli Diagnostics report figures of 90.9 and 98.8%. Christofi et al. (2002) set the sensitivity and specificity of the Bommeli Diagnostics ELISA at 83 and 98% in areas where the prevalence of E. granulosus infection in dogs was 0.2%, although the specificity was reduced to 80% in a group of dogs infected simultaneously with Taenia spp. The negative predictive value was >99.9%, but the positive predictive value only 7% (Christofi et al., 2002). The sensitivity of ELISA is closely linked to the worm burden of the infected individuals, being 29% in dogs harbouring less than 100 worms, but 92% in ones with more than 100 worms (Deplazes et al., 1994). Considering all strains of E. granulosus, the percentage of dogs infected may vary from 0.35 to 72.4% depending on the region (Eckert et al., 2001b). There are few previous reports on infection rates in dogs in areas affected by the sylvatic cycle of the parasite. Oksanen and Laaksonen (1995) examined 93 Finnish dogs in area 2 for taeniid eggs, with negative results, whereas Cameron (1960) found up to 25% of the dogs to be infected in many areas of north-western Canada, and Rausch (1995) refers to an unpublished Russian source according to which 5.6% of the dogs in eastern Siberia harboured E. granulosus. Even though the results of this study as well as all epidemiological data support the fact that E. granulosus has not spread to dog population in northern Finland, statistically the number of samples in the present study was too low to allow any definitive conclusion to be reached regarding the absence of infection. The size of the dog population was estimated by the local veterinarians to be 1000 dogs in the first sampling and 3000 dogs in the second. The negative results show only that the prevalence of infection is less than 1.6%, with a confidence level of 95%, assuming that the test sensitivity was 100%. Even though the Bommeli Diagnostics ELISA has not been validated for the examination of wolves, the kit seems to be a good choice for the screening of the northern biotype of E. granulosus. The finds of E. granulosus worms in wolves and their family groups by the sedimentation method suggest that the positive ELISA results are true positives, although there was one intestinal sample with a positive ELISA result that was negative when the sedimentation method was used. The reason for the inconsistent results may be a false positive result in ELISA, the cut-off value being too low for the examination of wolf populations, or a false negative result in the sedimentation test, because of a very low worm burden or deterioration of worms after the death of the animal. Cross-reactions caused by other helminth infections similarly cannot be excluded.
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Wolves infected with E. granulosus spread eggs around their territories. However, wolves tend to avoid human settlements. The risk to human health caused by infected wolves through possible contamination of berries and mushrooms has been evaluated as being minimal in Finland (Maijala et al., 2002). Hydatid disease in humans caused by the northern biotype has been reported to be benign compared with that caused by strains from domestic cycles, which can cause a highly symptomatic, hazardous disease requiring surgical treatment (Wilson et al., 1968). Population studies during the 1990s using various imaging techniques, including ultrasonography, have nevertheless shown that up to 60% of the cystic echinococcosis cases may also be asymptomatic (Pawlowski et al., 2001). The possible benign manifestation of the Scandinavian biotype of E. granulosus should be re-evaluated in view of the later findings reviewed by Pawlowski et al. (2001). Because dogs are considered the most important source of human infections, measures to control hydatid disease should be focused on the prevention and treatment of E. granulosus infections in dogs.
5. Conclusions E. granulosus occurs in eastern Finland in a sylvatic cycle involving wolves as the definitive host and reindeer, and probably also elk and forest reindeer as intermediate hosts. The reindeer seems to be a good sentinel animal to show the contamination of soil with Echinococcus eggs. The results of both this study and a previous work indicate that E. granulosus infection has not spread to dogs in the reindeer herding area, but because dogs are considered the most important source of human infections, measures to control hydatid disease should be focused on the prevention of E. granulosus infections in dogs.
Acknowledgements We thank local veterinarians, Pirjo Korhonen and Sauli Laaksonen, for good co-operation in organising the collecting of dog samples. We also thank the staff of EELA for excellent technical work. This work was supported by a grant from the Finnish Foundation for Veterinary Medicine.
References Analytical Software, 2000. Statistix for Windows, Version 7 (Manual), Analytical Software, Tallahassee, FL, USA, 359 pp. Arnesen, K., 1953. Echinokokkcyste i lunge. Tidskr. Norske laegefor. 73, 132–134. Brüning, J., 1948. Echinococcus. Sv. Vet. Tidskr. 53, 219–223. Cameron, T.W.M., 1960. The incidence and diagnosis of hydatid cyst in Canada. Echinococcus granulosus var. canadensis. Parasitologia II (3), 381–390. Cederberg, O.-E., 1946. Beiträge zur Kenntnis über das Vorkommen von Echinococcus-fällen in Finnland. Acta Chir. Scand. 93, 111–130.
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Christofi, G., Deplazes, P., Christofi, N., Tanner, I., Economides, P., Eckert, J., 2002. Screening of dogs for Echinococcus granulosus coproantigen in a low endemic situation in Cyprus. Vet. Parasitol. 104, 299–306. Deplazes, P., Gottstein, B., Eckert, J., Jenkins, D.J., Ewald, D., Jimenez Palacios, S., 1992. Detection of Echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs, dingoes and foxes. Parasitol. Res. 78, 303–308. Deplazes, P., Jimenez-Palacios, S., Gottstein, B., Skaggs, J., Eckert, J., 1994. Detection of Echinococcus coproantigens in stray dogs of northern Spain. Appl. Parasitol. 35, 297–301. Eckert, J., Gemmell, M.A., Matyas, Z., Soulsby, E.J.L. (Eds.), 1984. WHO Guidelines for Surveillance, Prevention and Control of Echinococcosis/Hydatidosis. World Health Organization, Geneva, 148 pp. Eckert, J., Deplazes, P., Craig, P.S., Gemmell, M.A., Gottstein, B., Heath, D., Jenkins, D.J., Kamiya, M., Lightowlers, M., 2001a. Echinococcosis in animals: clinical aspects, diagnosis and treatment. In: Eckert, J., Gemmell, M.A., Meslin, F.-X., Pawlowski, Z.S. (Eds.), WHO/OIE Manual on Echinococcosis in Humans and Animals: A Public Health Problem of Global Concern. World Organisation for Animal Health and World Health Organization, Paris, pp. 72–99. Eckert, J., Schantz, P.M., Gasser, R.B., Torgerson, P.R., Bessonov, A.S., Movsessian, S.O., Thakur, A., Grimm, F., Nikogossian, M.A., 2001b. Geographic distribution and prevalence. In: Eckert, J., Gemmell, M.A., Meslin, F.-X., Pawlowski, Z.S. (Eds.), WHO/OIE Manual on Echinococcosis in Humans and Animals: A Public Health Problem of Global Concern. World Organisation for Animal Health and World Health Organization, Paris, pp. 100–142. Huldt, G., Johansson, S.G.O., Lantto, S., 1973. Echinococcosis in northern Scandinavia. Arch. Environ. Health 26, 36–40. Kojola, I., 2000. Suurpetojen lukumäärä ja lisääntyminen vuonna 1999. Riistantutkimuksen Tiedote, vol. 165. Finnish Game and Fisheries Research Institute, Helsinki, pp. 1–8. Kojola, I., 2001. Suurpetojen lukumäärä ja lisääntyminen vuonna 2000. Riistantutkimuksen Tiedote, vol. 175. Finnish Game and Fisheries Research Institute, Helsinki, pp. 1–6. Kummeneje, K., 1982. Bekjempelse av ekinokokkose (hydatidose) hos rein og hund. Status slaktesesongen 1981/82 ved Kautokeino Slakteri. Norsk Veterinaertidsskrift 94, 6. Lindholm, Å., Lantto, S., 1968. Hydatid disease in Northern Sweden. Arch. Environ. Health 17, 685–688. Maijala, R., Haukisalmi, V., Henttonen, H., Hirvelä-Koski, V., Kauhala, K., Kilpelä, S.-S., Meri, T., Nylund, M., Tenhu, H., Saari, S., 2001. Risk Assessment of the Spread of Echinococcus multilocularis into and within Finland. National Veterinary and Food Research Institute (EELA), Helsinki. Maijala, R., Haukisalmi, V., Henttonen, H., Hirvelä-Koski, V., Kauhala, K., Kilpelä, S.-S., Lavikainen, A., Oksanen, A., Tenhu, H., Vahteristo, L., 2002. Risk Assessment on Echinococcus granulosus in Finland. National Veterinary and Food Research Institute (EELA), Helsinki. Myrseth, O., 1956. Echinokokksykdommen i Finnmark. Tidskr. Norske laegefor. 76, 867–871. Oksanen, A., Laaksonen, S., 1995. Ekinokokin jälkilypsy: kuusamon koirien suuri ulostetutkimus talvella 1993. Suom. Eläinlääkär. 101, 168–172. Pawlowski, Z.S., Eckert, J., Vuitton, D.A., Ammann, R.W., Kern, P., Craig, P.S., Dar, K.F., De rosa, F., Filice, C., Gottstein, B., Grimm, F., Macpherson, C.N.L., Sato, N., Todorov, T., Uchino, J., von sinner, W., Wen, H., 2001. Echinococcosis in humans: clinical aspects, diagnosis and treatment. In: Eckert, J., Gemmell, M.A., Meslin, F.-X., Pawlowski, Z.S. (Eds.), WHO/OIE Manual on Echinococcosis in Humans and Animals: A Public Health Problem of Global Concern. World Organisation for Animal Health and World Health Organization, Paris, pp. 20–71. Pöysti, H., Pöysti, E., 1969. Havaintoja ekinokokkoosista (E. granulosus) poroissa (observations on echinococcosis (E. granulosus) in reindeer). Eläinlääkärilehti 75, 135–137. Rausch, R., 1956. Studies on the helminth fauna of Alaska. The occurrence of Echinococcus multilocularis Leuckart, 1863, on the mainland of Alaska. Am. J. Trop. Med. Hyg. 5, 1086–1092. Rausch, R.L., 1995. Life cycle patterns and geographic distribution of Echinococcus species. In: Thompson, R.C.A., Lymbery, A.J. (Eds.), Echinococcus and Hydatid Disease. CAB International, Wallingford, pp. 89–134. Rein, K., 1957. Echinokokksykdommen i Nord-Norge. Nord. Med. 28, 1853–1857. Roneus, O., 1974. Prevalence of echinococcosis in reindeer (Rangifer tarandus) in Sweden. Acta Vet. Scand. 15, 170–178. Skjenneberg, S., 1959. Ekinokokkose hos rein i Kautokeino. Nord. Vet. Med. 11, 110–123.
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Sweatman, G.K., Williams, R.J., 1963. Comparative studies on the biology and morphology of Echinococcus granulosus from domestic livestock, moose and reindeer. Parasitology 53, 339–390. Söderhjelm, L., 1946. Echinococcus Hydatidosus Hos Rein (Rangifer tarandus). Skand. Vet. Tidskr. 36, 378–381. Thomas, D.C., 1996. Prevalence of Echinococcus granulosus and Taenia hydatigena in caribou in north-central Canada. Rangifer (special issue no. 9), 331–336. Thompson, R.C.A., 1995. Biology and systematics of Echinococcus. In: Thompson, R.C.A., Lymbery, A.J. (Eds.), Echinococcus and Hydatic Disease. CAB International, Wallingford, pp. 1–50. Wilson, J.F., Diddams, A.C., Rausch, R.L., 1968. Cystic hydatid disease in Alaska. Am. Rev. Resp. Dis. 98, 1–15.
Echinococcus granulosus in Finland Varpu Hirvelä-Koski a,∗ , Voitto Haukisalmi b , Seija-Sisko Kilpelä c , Minna Nylund a , Perttu Koski a a
b
National Veterinary and Food Research Institute (EELA), Oulu Regional Unit, P.O. Box 517, FIN-90101 Oulu, Finland Finnish Forest Research Institute, Vantaa Research Center, P.O. Box 18, FIN-01301 Vantaa, Finland c Finnish Game and Fisheries Research Institute, Taivalkoski Game and Fisheries Research, Ohtaojantie 19, FIN-93400 Taivalkoski, Finland Received 20 June 2002; received in revised form 15 October 2002; accepted 16 October 2002
Abstract Echinococcus granulosus is shown to occur in eastern Finland in a sylvatic cycle involving wolves (Canis lupus) as the definitive host and reindeer (Rangifer tarandus tarandus) and probably also elk (Alces alces) and the wild forest reindeer (Rangifer tarandus fennicus) as intermediate hosts. Even though the prevalence of E. granulosus in reindeer has increased in recent years, it is still very low (<0.013%). The results suggest, however, that the reindeer is a good sentinel animal to show the contamination of soil with Echinococcus eggs. This is the first report of wolves acting as a definitive host for E. granulosus in northern Europe. The parasite seems to be quite common in the Finnish wolf population, with a prevalence of approximately 30% in both intestinal and faecal samples. The present results and previous ones indicate that E. granulosus infection has not spread to dogs in the reindeer herding area. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Echinococcus granulosus; Cestoda; Reindeer; Wolf; Dog; Finland
1. Introduction Echinococcus granulosus has occurred earlier as an endemic infection in reindeer herding area in the northern parts of Fennoscandia, where the life-cycle involved the dog as the definitive host and the reindeer (Rangifer tarandus tarandus) as the intermediate host (Skjenneberg, 1959; Pöysti and Pöysti, 1969; Roneus, 1974; Kummeneje, 1982). The prevalence in reindeer varied between regions, the highest values being 10–14% (Skjenneberg, ∗ Corresponding author. Tel.: +358-8-5622-633; fax: +358-8-5544-977. E-mail address: [email protected] (V. Hirvelä-Koski).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 3 8 1 - 3
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1959; Söderhjelm, 1946). There are no reports on the rate of infection of dogs in this area, however, E. granulosus infections have occasionally been found in humans, mostly in Lapps living in close contact with their dogs, which were used for shepherding the reindeer (Söderhjelm, 1946; Cederberg, 1946; Brüning, 1948; Arnesen, 1953; Myrseth, 1956; Rein, 1957; Lindholm and Lantto, 1968; Huldt et al., 1973). However, in Finland no domestic incidences of E. granulosus infection in humans have been encountered for several decades (Maijala et al., 2002). Circumstances in northern Finland have changed since the 1960s, due to obligatory inspection of all reindeer meat since 1975, better hygiene in handling the slaughter offal and a higher standard of living among the reindeer owners. The use of shepherd dogs has also diminished since snowmobiles were adopted for rounding up the reindeer herds from the 1960s onwards. However, since 1992 E. granulosus cysts have again been regularly found in reindeer carcasses in connection with meat inspections. It is not known whether this is due to a true increase in its prevalence or to increased efficiency of meat inspection. The first abattoir for reindeer slaughtering was built in 1984, but it was not until 2001 that all reindeer to be slaughtered were transported to abattoirs and inspected there. Hydatid cysts have been found both at abattoirs and at slaughtering places in the field. The occurrence of echinococcosis in livestock in Finland is monitored in connection with meat inspection. Hydatid cysts have occasionally been found in horses and in one cow (National Veterinary and Food Research Institute, Finland, unpublished data), but never in sheep, goats or pigs. Echinococcus multilocularis has never been found in Finland (Maijala et al., 2001). We describe here the occurrence of hydatid cysts in reindeer in Finland and report on the results of examinations of potential definitive hosts for E. granulosus, i.e. dogs and wolves. 2. Materials and methods 2.1. Areas studied Areas 1, 2 and 3 are situated along the eastern border of Finland (Fig. 1). Other parts of the country are designated as area 4. Areas 1 and 2 are in the reindeer herding area. Area 1 is located between 27◦ 30 and 29◦ 30 E and 70◦ and 68◦ 30 N, area 7586 km2 consisting half of the municipality of Inari, population density being 0.5 inhabitants/km2 . Area 2 consists of municipalities Salla, Suomussalmi, Hyrynsalmi and Kuusamo, located between 28◦ and 30◦ 30 E and between 67◦ 45 and 64◦ 30 N, area 17 445 km2 and the population density ranging from 0.9 to 3.5 inhabitants/km2 . Area 3 is located between 26◦ 30 and 31◦ 30 E and between 64◦ 30 and 60◦ 20 N, consisting of 28 municipalities, the area being 26 830 km2 and the population density 2.0–35.2 inhabitants/km2 in rural parts and up to 522.3 in the cities. The areas 1–3 all represent northern boreal forest. The forests in area 1 are dominated by pine (Pinus sylvestris), and in areas 2 and 3 by spruce (Picea abies) and pine. Birches (Betula pendula and B. pubescens) are the most common deciduous trees in all areas. The average temperature of July is ca. +10 ◦ C in area 1, between +10 and 15 ◦ C in area 2
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Fig. 1. Geographical distribution and numbers of wolf samples in 1999–2001. The first column: number of samples; the second column: number of positive samples. Symbols: ( ) dried faecal samples; ( ) frozen faecal samples; ( ) intestinal samples.
and ca. +15 ◦ C in area 3. The yearly precipitation varies between 500 and 700 mm, being lowest in area 1. In areas 1, 2 and 3 the length of the growing season is 100, 100–150 and 150–220 days and the length of snow cover 200–250, 150–200 and 100–150 days per year, respectively. 2.2. Examination of farm livestock and reindeer In Finnish slaughterhouses, the carcasses of semi-domesticated reindeer (R. tarandus tarandus) and farm livestock, including cattle, sheep, goat, horses and pigs, are routinely examined for Echinococcus metacestodes during meat inspection. When hydatid cysts are suspected, they are sent to the National Veterinary and Food Research Institute to be examined for echinococcosis in terms of gross pathology, microscopy and histology. These examinations provide descriptions of the appearance of the cysts and their elasticity, size, number and location and exclude the possibility of other parasitic infections or cysts of non-parasitic origin. The cyst contents and scrapings from the cyst wall are examined for Echinococcus protoscoleces of characteristic morphology by microscopy. For sterile and degenerated cysts, histological examination is performed using haematoxylin-eosin staining to show the typical structures of the cyst wall, with an outer acellular laminar
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hyaline layer and a thin syncytial germinal layer. The cyst is surrounded by a fibrous capsule and infiltrate of inflammatory cells, including giant cells and eosinophils. In the case of degenerated cysts, the diagnosis is confirmed by identification of the outer laminar layer. All information concerning the infected reindeer during 1979–2001 were obtained from the diagnostic data of National Veterinary and Food Research Institute. The numbers of reindeer slaughtered annually in 1979–2000 were obtained from the official statistics of the Association of Reindeer Owners. 2.3. Wolves 2.3.1. Dried faecal samples A total of 110 faecal samples were collected from areas 3 (n = 109) and 4 (n = 1) between March and October 1999 (Fig. 1) and kept on an open petri dish at room temperature until they were dry and their weight was stable. After examination for taeniid eggs by the flotation method, the samples were stored in a frozen state at –20 ◦ C. Thirty of them were examined for Echinococcus coproantigen by ELISA (Bommeli Diagnostics), the average time elapsing between collection and the flotation test being 86 days (range 47–268 days). 2.3.2. Frozen faecal samples A total of 332 faecal samples were collected from the field in the years 1999–2001. Nearly all of them (n = 330) were from area 3 and the rest (n = 2) from area 2. Most of the samples represented one animal, but there were also some pooled samples. In order to study the life habits of wolves, the alpha pair and some juvenile wolves in several packs had been radio-collared and followed by radiotelemetry (Kojola, 2002, personal communication). In 2001, for instance, there were 17 radio-collared wolves and five family groups under observation in this way. On the basis of this information the faecal samples could often be identified to a certain group of wolves and samples could be collected especially around the dens, from daily resting places after the wolves had left and from around prey animals 2–3 days after the kill. The age of the sample at the time of collection was estimated by the field worker. Before arriving at the laboratory most of the samples had been frozen for storage purposes, however, and the conditions of preservation in the field were not documented. In the laboratory the samples were kept frozen at −20 ◦ C until tested for Echinococcus coproantigen by ELISA (Bommeli Diagnostics). The average time elapsing between collection and arrival at the laboratory was 64 days (range 0–365 days), and that between arrival and ELISA testing 326 days (range 36–539 days). 2.3.3. Intestinal samples Intestinal samples were obtained from 23 wolves in 1999–2001. There were 12 males and 11 females. The wolves originated from areas 1, 2 and 3, near the eastern border of Finland (Fig. 1). Twenty of the wolves had been killed during hunting, two had been found dead in the field and one was shot after a traffic accident. In 11 cases the small intestine, large intestine and rectum were immersed in 5% formaline, in 11 cases they were frozen and in one case the whole carcass was frozen at −20 ◦ C (Table 3). The formaline-fixed samples were examined for Echinococcus worms by the sedimentation method, while the
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frozen samples were first examined for Echinococcus coproantigens with ELISA (Bommeli Diagnostics ), and those giving positive or ambiguous ELISA values were tested further by the sedimentation method. Seven of the frozen samples were also examined for taeniid eggs using the flotation test. 2.4. Dogs All the dog samples were collected from area 2, in the eastern part of the reindeer herding area (Fig. 1). Faecal samples were obtained from 352 dogs in co-operation with local veterinarians in the years 1999 and 2001. The sampling period extended from February to May in both years. Most of the samples (68%) were obtained from dogs used for hunting and five from ones that had been used as shepherd dogs in reindeer herding. Antiparasitic drugs had been given to 61% of the dogs during the year before sampling. About 20% of the dogs had been treated with drugs containing praziquantel. The fresh samples taken in 1999 (n = 169) were kept cool and sent to the laboratory once a week. After examination using the flotation test, they were kept frozen at −20 ◦ C until tested for Echinococcus coproantigen with ELISA (Genzyme Virotech GmbH). Samples giving positive or ambiguous results were re-tested with Bommeli Diagnostics ELISA. In 2001, the fresh samples (n = 183) were kept frozen at −20 ◦ C until examined for Echinococcus coproantigen with ELISA (Bommeli Diagnostics) and for taeniid eggs by the flotation method. Repeat samples were tested in the case of seven dogs. 2.5. Flotation method The faecal sample was homogenised by manual pressing in a plastic bag, 3.0 g was weighed out, diluted to 42 ml with tepid tap water, mixed thoroughly and strained through a tea sieve. After centrifugation of 12.5 ml of the filtrate (660 × g, 3 min), the supernatant was removed until there was approximately a 2 mm layer of fluid above the sediment. Concentration solution (NaCl 393 g, saccharose 50 g, water 1000 ml, specific gravity 1.21) was added to a total volume of 2.5 ml and mixed thoroughly. A Mc Master counting chamber was filled with the solution and the number of eggs was counted under low magnification. 2.6. Sedimentation method The sedimentation method was performed as described by Eckert et al. (1984). As a safety precaution, the frozen samples were moved to −70 ◦ C for 7 days before examination. After thawing, the small intestine was divided into 4–6 sections, each of which was split longitudinally and immersed in physiological saline at 37 ◦ C. After 30 min, the intestinal wall was scraped and the scrapings were added to the solution. After sieving (mesh size 1.5 mm), the solution was poured into a high vessel and left to settle for 30 min. The supernatant was removed with a pipette. The sedimentation was repeated several times to remove the flocculant material. A small amount of the sediment at a time was poured into a black tray and examined for adult Echinococcus worms or their fragments under a stereo microscope. The number of worms was determined by the number of Echinococcus scoleces found.
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For specific identification of Echinococcus, several entire or fragmented strobilae from two wolves were stained with Semichon’s acetic carmine, cleared in eugenol and mounted in Canada balsam. 2.7. ELISA for the detection of Echinococcus-specific coproantigens Two commercial kits were used for the detection of Echinococcus-specific coproantigens, Echinococcus ELISA (Genzyme Virotech GmbH, Rüsselsheim, Germany) and Echinotest (Bommeli Diagnostics, Liebefeld-Bern, Switzerland). The tests were performed according to the manufacturers’ instructions. All faecal samples were frozen for 7 days at −70 ◦ C before examination. In the case of frozen intestines, the sample was taken either from the rectum or from the posteriormost part of the large intestine. All samples were tested in duplicate. In Genzyme Virotech ELISA, values <9.0 were interpreted as negative, 9.0–11.0 as ambiguous and >11.0 as positive, while in Bommeli Diagnostics ELISA values below 30 were regarded as negative, 30–40 as ambiguous and values exceeding 40 as positive. If the ELISA result was positive or ambiguous, the test was repeated once or twice, the values for the different test runs being averaged to obtain the final result. 2.8. Statistics The underlying assumptions for the parametric tests were often invalid because the data were asymmetrical or not normally distributed, and non-parametric tests were therefore used. The Mann–Whitney U-test was performed to compare the time interval from collection to freezing between the wolf samples with positive and negative ELISA values. Spearman’s rank correlation was used to test for an association between the estimated ages of the wolf samples and their corresponding ELISA values. The number of infected reindeer among the adult animals slaughtered in 1992–1995 in area 2 was compared with the situation in 1996–1999 by means of the χ2 -test, which was also used to test the differences in the proportion of infected wolves in five family groups. Statistical analyses were carried out using the analytical software package Statistix for Windows (Analytical Software, 2000).
3. Results 3.1. Prevalence of E. granulosus in farm animals and reindeer E. granulosus cysts have been found in Finland during the years 1979–2001 in five horses and one cow (unpublished data from National Veterinary and Food Research Institute, Finland). The positive horses found in 1998 and 1999 were from area 4 and the cow, found in 1986, from area 3 (Fig. 1). The geographical origins of the positive horses found in 1979, 1992 and 1994 are unknown. Echinococcosis was reported in one reindeer during the period of 13 years from 1979 to 1991 (Table 1), but cases have been diagnosed annually since 1992, and a total of 59 reindeer had been shown to have been infected with hydatid cysts by 2001. The proportion of infected animals in the whole population of slaughtered reindeer ranged between 0.008
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Table 1 Number and proportion of reindeer infected with Echinococcus granulosus in all slaughtered reindeer and among adult animals (>1 year of age) in Finland in 1979–2001 Year(s)
Number of reindeer infected
Infections per 1000 reindeer
Infections per 1000 adults
Number of slaughtered adults
1979–1986 1987 1988–1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001
0 1 0 1 1 1 4 1 10 10 12 6 13
– – – 0.008 0.008 0.008 0.033 0.012 0.111 0.104 0.129 0.069 –
– – – 0.023 0.026 0.030 0.118 0.037 0.391 0.382 0.453 0.233 –
– – – 42 751 37 892 33 397 33 938 27 369 25 586 26 156 26 479 25 781 –
Statistics on the numbers of reindeer slaughtered are not yet available for 2001.
and 0.129 per 1000 animals yearly, and that among adult animals (>1 year) between 0.023 and 0.453 per 1000 (Table 1). In both groups, there seems to have been more than 10-fold increase in the proportion between 1994 and 1997. The positive animals originated mostly from eastern border, close to Russia, except for one case from the western part of the reindeer herding area in the year 1999 (Fig. 2). During the period 1992–1995 there were 7 reindeer with hydatid cysts out of a total of 17 995 adult animals slaughtered in area 2, the corresponding figures in 1996–1999 being 32 out of 12 434. The χ2 -test showed a statistically significant increase in the number of infected adult animals found in connection with meat inspection between the two periods (P < 0.001). There were at least two distinct clusters in the occurrence of hydatid cysts in area 2, the proportion of positive cases in the more northerly one in 1995–1999 being 2.1 per 1000 reindeer slaughtered or 7.5 per 1000 adult reindeer slaughtered. Information on age and sex was obtained with respect to 27 infected reindeer, including 23 adult females, 2 adult males and 2 juveniles (less than 1 year old). The exact age was known in 12 cases, 25% being 3–7 years old and 75% 9–11 years old. The site of the cysts was most often in the lung (89.8%), but the liver and occasionally the spleen were also involved (liver 5.1%, liver and lung 3.4%, spleen and lung 1.7%). The number of cysts detected per animal varied between one and seven and the diameter between 0.5 and 10 cm. Most of the animals (52.5%) harboured one cyst, 23.7% two cysts, 15.3% three cysts, 6.8% four cysts and 1.7% seven cysts. Degenerated or calcified cysts were detected in six reindeer (10%). In 45 reindeer (76%), the cysts were fertile and direct microscopy of the contents revealed E. granulosus protoscoleces. The diameter of the fertile cysts was 3–8 cm in 31 cases (69%) and more than 8 cm in 13 cases (29%). Only one of the fertile cysts (2%) was less than 3 cm in diameter. In 14 cases, no protoscoleces were found and the diagnosis was based on histological examination. The sterile cysts were smaller, their diameter being under 3 cm in 77% of the cases, although the largest again measured 10 cm.
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Fig. 2. Geographical distribution of echinococcosis detected in Finnish reindeer during meat inspection in 1992–2001. Each dot marks one infected reindeer (n = 58).
3.2. Wolves 3.2.1. Dried faecal samples All the dried faecal samples collected in 1999 were negative for taeniid eggs in the flotation test, but eight contained Toxocara sp. eggs 20–1200 EPG. Three of the 30 samples tested with ELISA gave positive results, with values of 128, 75 and 73. Flotation testing of the positive samples did not reveal any signs of parasitic infections. The time between collection and freezing had been 73–78 days, and that between freezing and testing 494–577 days. 3.2.2. Frozen faecal samples The distribution of ELISA values for the frozen faecal samples is shown in Fig. 3. A total of 96 of the samples (29%) were positive in the ELISA test, with values ranging from 40 to
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Fig. 3. Distribution of ELISA values for faecal samples from wolves (n = 332) and dogs (n = 183) tested for Echinococcus coproantigen by Bommeli Diagnostics ELISA.
242. There were 13 samples (4%) giving ambiguous values between 30 and 40, while most of the negative samples (86%) gave values below 21. ELISA results for six family groups (packs) are presented in Table 2. All the samples for packs 3 and 6 were negative in ELISA. The number of samples examined in the negative packs was nevertheless too small to allow any definitive conclusion to be reached regarding the absence of E. granulosus infection in these groups, but the χ2 -test result did indicate a slight difference between the groups (0.05 < P < 0.1). The temporal distribution of the samples from groups 1, 2 and 5 is presented in Fig. 4. No seasonal variation could be observed in the prevalence of E. granulosus. One wolf in group 1, two in group 2 and one in group 3 had been killed during 2001 (Table 3, numbers 9, 13, 14 and 20, respectively). In two of them (in groups 1 and 2) E. granulosus was detected in the intestine by sedimentation method, the approximate numbers of worms being 110 and 1670, respectively. All the samples giving ELISA values >30 were re-tested. Of the 95 samples positive in the first test run, 92 (97%) remained positive after the second run, while three samples with low ELISA values (43, 44 and 47) gave negative or ambiguous values in the second run (29, 19 and 30, respectively). Seventeen samples gave ambiguous ELISA values in the first test run, six of which became negative in the second run, six still gave ambiguous values and five became positive. The age of the faecal mass at the time of collection was assessed by the collector. Most of the samples were estimated to be less than 8 days old, the variation being from <1 day to 1–2 months. There was no significant correlation between the ELISA value and the age among these 0–7 days old samples (Spearman’s rank correlation coefficient −0.023, n = 225, P > 0.05). The oldest positive samples were estimated to be 10–14 days and 1–2 months old, with ELISA values of 185 and 76, respectively. The median time elapsing between collection and freezing in the laboratory was 53 days (range 4–365 days) for the samples with a positive ELISA result and 49.5 days (range 0–325 days) for the negative samples.
184
Pack number
Collecting period
1 2 3 4 5 6
January 2000 to May 2001 September 1999 to May 2001 May 2000 to December 2000 January 2001 to July 2001 May 2001 to September 2001 August 2000 to September 2001
Total
Number of samples
Number of positive samples
Percentage of positive samples
71 98 6 2 42 6
27 36 0 2 11 0
38 37 0 100 26 0
225
76
34
Number of ambiguous results
Percentage of ambiguous results
Range of negative ELISA values
Range of positive ELISA values
7 2 0 0 1 0
10 2 0 0 2 0
0–27 0–29 2–23 – 0–21 0–29
44–213 43–242 – 92–112 43–129 –
10
4
–
–
All samples giving positive or ambiguous test results were re-tested once or twice and the values for the different test runs were averaged to obtain the final result.
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Table 2 ELISA (Bommeli Diagnostics) results for six family groups (packs) of wolves
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Fig. 4. Seasonal variations in the number of faecal samples and ELISA results for three family groups (packs) of wolves in 2000–2001.
This difference was not statistically significant in the Mann–Whitney U-test (U = 12 808, n = 323, P > 0.05). 3.2.3. Intestinal samples The results of the examinations of the intestinal samples are shown in Table 3 and Fig. 1. Echinococcus worms were found by the sedimentation method in six wolves, while eight
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Table 3 Results of intestinal samples from wolves Date of death
Area
Preservation of the intestine
Sedimentation method
Approximate number of worms
Identification of E. granulosus
ELISA result (%)
Flotation method
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
20.11.1999 10.2.2000 27.10.2000 27.10.2000 30.10.2000 31.10.2000 16.1.2001 19.1.2001 19.1.2001 23.1.2001 25.1.2001 25.1.2001 25.1.2001 25.1.2001 26.1.2001 28.2.2001 2.3.2001 3.3.2001 5.3.2001 9.3.2001 15.3.2001 31.5.2001 2.6.2001
2 3 2 2 3 1 3 3 3 3 2 2 2 2 2 2 2 2 3 3 3 3 3
Formaline-fixed Formaline-fixed Formaline-fixed Formaline-fixed Formaline-fixed Formaline-fixed Formaline-fixed Formaline-fixed Formaline-fixed Frozen Formaline-fixed Frozen Frozen Frozen Formaline-fixed Frozen Frozen Frozen Frozen Frozen Frozen Frozen Frozen
Positive Positive Negative Negative Negative Negative Negative Positive Positive ND Negative ND Negative Positive Negative ND ND ND ND ND ND ND Positive
5 364 0 0 0 0 0 5 110 – 0 – 0 1670 0 – – – – – – – 718
× × – – – – – – – – – – – – – – – – – – – – –
ND ND ND ND ND ND ND ND ND 12 ND 6 76 119 ND 16 6 8 0 11 0 14 115
ND ND ND ND ND ND ND ND ND Negative ND ND ND Negative ND Negative Negative ND Negative ND Negative ND Negative
Bommeli Diagnostics ELISA was used for the detection of Echinococcus coproantigen.
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were negative, the positive wolves having been killed in areas 2 and 3. Because of the abundance of flocculent material in the sediment in spite of several washings, only a small amount of the sediment could be examined under the microscope at a time, which meant that only 20% of the sediment was examined in case of the negative samples. The proportion of the sediment examined in the case of the positive samples varied between 10 and 50%. The number of worms was determined by relating the number counted to the volume of sediment examined. The estimated number of worms per infected wolf varied between 5 and 1670. Three of the frozen samples were positive in ELISA, giving values of 76, 115 and 119, but in one of these, with an ELISA value of 76, no worms could be detected by the sedimentation method even though the whole sediment in this sample was examined. Nine samples were negative in the ELISA test, with values ranging between 0 and 16, and there were seven samples examined by the flotation method in which no taeniid eggs were found. The stained and mounted adult specimens of Echinococcus from wolves usually lacked rostellar hooks, but their other morphological features, particularly the position of the genital pore in mature segments (at the mid-level), and the number (ca. 50) and distribution of testes (anterior and posterior to the level of the genital pore) showed unambiguously that these specimens represented E. granulosus, rather than E. multilocularis (see Rausch, 1956 and Thompson, 1995 for the identification of Echinococcus species). 3.3. Dogs All the faecal samples from dogs were negative for taeniid eggs in the flotation test in both years. The first sampling in 1999 (n = 169) included six positive samples in ELISA and two with an ambiguous result, but they all became negative in the second test run. The ELISA values varied between 9.9 and 28.6 (Genzyme Virotech) in the first run and between 0 and 25 in the second (Bommeli Diagnostics). Although the ELISA values of the negative samples varied from 0 to 8.8, most of the negative samples (152/161) gave values lower than five with the Genzyme Virotech test. Ten samples out of the 183 taken in 2001 (6%) were positive and 7 (4%) gave ambiguous values based on the averaged results of the two test runs with Bommeli Diagnostics ELISA. The distribution of ELISA values is shown in Fig. 3. The negative values varied between 0 and 29 and the positive ones between 43 and 62. Of the 12 samples giving ambiguous values in the first test run, 6 became negative in the second run, 3 remained ambiguous and 3 became positive. Of the seven positive samples, six remained positive and one became ambiguous in the second run. No taeniid eggs were found in any of the samples by the flotation method, nor did this test reveal any other parasitic infections in the dogs gaining false positive or ambiguous values in ELISA. Repeated samples obtained from seven dogs with positive ELISA values varying between 35 and 62 in the first sampling were all negative, with ELISA values ranging from 4 to 26. The time elapsing between the two samplings was 25–43 days. All except one of the dogs initially giving a positive value were used for hunting, and only one of these had been given antiparasitic drugs effective against Echinococcus within a year of sampling.
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4. Discussion This is the first report of wolves acting as a definitive host for E. granulosus in northern Europe. The sylvatic life-cycle in Finland involves wolves and cervids, i.e. reindeer, and most probably also the elk (Alces alces) and the wild forest reindeer (Rangifer tarandus fennicus), which are the main prey species for wolves in eastern Finland. The parasite seems to be quite common in the Finnish wolf population, the results for both the intestinal and faecal samples indicating a prevalence of approximately 30%. This figure is comparable to the infection rates of wolves reported from North America and the Russian far east, ranging from 14 to 72% (Rausch, 1995). There was some clustering among the family groups, probably as a natural consequence of the custom of wolves to prey as a pack (Table 2). There was no clear seasonal variation in the occurrence of E. granulosus in wolves (Fig. 4). The minimum population of wolves in Finland was 98 animals in 1999, 130 in 2000 and 120 in 2001 (Kojola, 2000, 2001). The occurrence of E. granulosus in livestock throughout Finland has been monitored in accordance with the meat inspection instructions since the 1970s. Hydatid cysts have been occasionally found in horses and in one cow, but never in sheep or pigs, even though there are farms rearing both sheep and cattle even in areas 1, 2 and 3. In the case of the positive cow found in area 3 in 1986, wolves were suspected as the possible origin of the infection. The horse infections seem to have no epidemiological connection with infections by the Scandinavian cervid strain. The present figures describing the infection rate in reindeer are very low compared with those reported for other regions where the northern biotype of E. granulosus occurs in the sylvatic cycle. In parts of Canada, where the wolf is the chief natural primary host, 9–15.5% of the reindeer harboured hydatid cysts (Cameron, 1960), while Thomas (1996) reported that 2% of the caribou in the 2–5 years class and 8% of the older individuals were infected. Rausch (1995) reported prevalences of 3 and 5% in wild reindeer in arctic Alaska. In elk or moose (A. alces), prevalences of the northern biotype ranging from 24 to 59% have been reported in North America, and a figure as high as 71% in the far east of Russia (Rausch, 1995). In Fennoscandia, the prevalence in semi-domesticated reindeer has been 1.2–1.6% (Pöysti and Pöysti, 1969; Roneus, 1974; Kummeneje, 1982), higher figures can be found in older reports (9.6% in Norway, Skjenneberg, 1959). There are no permanent family groups of wolves in the reindeer herding area in Finland, and single wolves moving about in the area are killed rapidly in order to avoid reindeer losses. The absence of a permanent high-density population of the definitive host probably explains the low infection prevalences in reindeer in Finland. The reindeer pasture most of the year free in the forests and marshlands feeding on grass, sedge, mushrooms, lichen and other plants growing low near the ground. Because of its feeding habits, semi-domesticated reindeer seems to be a good sentinel animal to show contamination of earth and plants with eggs of the northern biotype of E. granulosus. In addition, hydatidosis in reindeer can easily be detected and diagnosed via the meat inspection process performed by the local veterinarians. Dogs can also serve as a definitive host for the northern biotype of E. granulosus (Cameron, 1960; Sweatman and Williams, 1963; Wilson et al., 1968; Rausch, 1995), and they are considered the main source of human infections in endemic areas where there is a
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sylvatic life-cycle of E. granulosus with wolves as the primary definitive hosts (Cameron, 1960; Wilson et al., 1968). Dogs have been regarded as the source in all reports on human infections in the northern parts of Fennoscandia (Söderhjelm, 1946; Cederberg, 1946; Brüning, 1948; Arnesen, 1953; Myrseth, 1956; Rein, 1957; Lindholm and Lantto, 1968; Huldt et al., 1973). According to the present results, E. granulosus has not spread to dogs. Dog samples from area 3 (Fig. 1) should also be examined, however, to confirm the absence of E. granulosus in dogs. Coproantigen detection by enzyme-linked immunosorbent assay (ELISA) has been deemed a practical method for screening E. granulosus in the definitive host because of its high sensitivity and specificity and high probability of correlation with current infection (Eckert et al., 2001a). ELISA detects infected animals within the pre-patent period and converts to negative in a few days after praziquantel treatment (Deplazes et al., 1992). Genzyme Virotech place the sensitivity and specificity of their ELISA test at 76 and 95%, respectively, while Bommeli Diagnostics report figures of 90.9 and 98.8%. Christofi et al. (2002) set the sensitivity and specificity of the Bommeli Diagnostics ELISA at 83 and 98% in areas where the prevalence of E. granulosus infection in dogs was 0.2%, although the specificity was reduced to 80% in a group of dogs infected simultaneously with Taenia spp. The negative predictive value was >99.9%, but the positive predictive value only 7% (Christofi et al., 2002). The sensitivity of ELISA is closely linked to the worm burden of the infected individuals, being 29% in dogs harbouring less than 100 worms, but 92% in ones with more than 100 worms (Deplazes et al., 1994). Considering all strains of E. granulosus, the percentage of dogs infected may vary from 0.35 to 72.4% depending on the region (Eckert et al., 2001b). There are few previous reports on infection rates in dogs in areas affected by the sylvatic cycle of the parasite. Oksanen and Laaksonen (1995) examined 93 Finnish dogs in area 2 for taeniid eggs, with negative results, whereas Cameron (1960) found up to 25% of the dogs to be infected in many areas of north-western Canada, and Rausch (1995) refers to an unpublished Russian source according to which 5.6% of the dogs in eastern Siberia harboured E. granulosus. Even though the results of this study as well as all epidemiological data support the fact that E. granulosus has not spread to dog population in northern Finland, statistically the number of samples in the present study was too low to allow any definitive conclusion to be reached regarding the absence of infection. The size of the dog population was estimated by the local veterinarians to be 1000 dogs in the first sampling and 3000 dogs in the second. The negative results show only that the prevalence of infection is less than 1.6%, with a confidence level of 95%, assuming that the test sensitivity was 100%. Even though the Bommeli Diagnostics ELISA has not been validated for the examination of wolves, the kit seems to be a good choice for the screening of the northern biotype of E. granulosus. The finds of E. granulosus worms in wolves and their family groups by the sedimentation method suggest that the positive ELISA results are true positives, although there was one intestinal sample with a positive ELISA result that was negative when the sedimentation method was used. The reason for the inconsistent results may be a false positive result in ELISA, the cut-off value being too low for the examination of wolf populations, or a false negative result in the sedimentation test, because of a very low worm burden or deterioration of worms after the death of the animal. Cross-reactions caused by other helminth infections similarly cannot be excluded.
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Wolves infected with E. granulosus spread eggs around their territories. However, wolves tend to avoid human settlements. The risk to human health caused by infected wolves through possible contamination of berries and mushrooms has been evaluated as being minimal in Finland (Maijala et al., 2002). Hydatid disease in humans caused by the northern biotype has been reported to be benign compared with that caused by strains from domestic cycles, which can cause a highly symptomatic, hazardous disease requiring surgical treatment (Wilson et al., 1968). Population studies during the 1990s using various imaging techniques, including ultrasonography, have nevertheless shown that up to 60% of the cystic echinococcosis cases may also be asymptomatic (Pawlowski et al., 2001). The possible benign manifestation of the Scandinavian biotype of E. granulosus should be re-evaluated in view of the later findings reviewed by Pawlowski et al. (2001). Because dogs are considered the most important source of human infections, measures to control hydatid disease should be focused on the prevention and treatment of E. granulosus infections in dogs.
5. Conclusions E. granulosus occurs in eastern Finland in a sylvatic cycle involving wolves as the definitive host and reindeer, and probably also elk and forest reindeer as intermediate hosts. The reindeer seems to be a good sentinel animal to show the contamination of soil with Echinococcus eggs. The results of both this study and a previous work indicate that E. granulosus infection has not spread to dogs in the reindeer herding area, but because dogs are considered the most important source of human infections, measures to control hydatid disease should be focused on the prevention of E. granulosus infections in dogs.
Acknowledgements We thank local veterinarians, Pirjo Korhonen and Sauli Laaksonen, for good co-operation in organising the collecting of dog samples. We also thank the staff of EELA for excellent technical work. This work was supported by a grant from the Finnish Foundation for Veterinary Medicine.
References Analytical Software, 2000. Statistix for Windows, Version 7 (Manual), Analytical Software, Tallahassee, FL, USA, 359 pp. Arnesen, K., 1953. Echinokokkcyste i lunge. Tidskr. Norske laegefor. 73, 132–134. Brüning, J., 1948. Echinococcus. Sv. Vet. Tidskr. 53, 219–223. Cameron, T.W.M., 1960. The incidence and diagnosis of hydatid cyst in Canada. Echinococcus granulosus var. canadensis. Parasitologia II (3), 381–390. Cederberg, O.-E., 1946. Beiträge zur Kenntnis über das Vorkommen von Echinococcus-fällen in Finnland. Acta Chir. Scand. 93, 111–130.
V. Hirvelä-Koski et al. / Veterinary Parasitology 111 (2003) 175–192
191
Christofi, G., Deplazes, P., Christofi, N., Tanner, I., Economides, P., Eckert, J., 2002. Screening of dogs for Echinococcus granulosus coproantigen in a low endemic situation in Cyprus. Vet. Parasitol. 104, 299–306. Deplazes, P., Gottstein, B., Eckert, J., Jenkins, D.J., Ewald, D., Jimenez Palacios, S., 1992. Detection of Echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs, dingoes and foxes. Parasitol. Res. 78, 303–308. Deplazes, P., Jimenez-Palacios, S., Gottstein, B., Skaggs, J., Eckert, J., 1994. Detection of Echinococcus coproantigens in stray dogs of northern Spain. Appl. Parasitol. 35, 297–301. Eckert, J., Gemmell, M.A., Matyas, Z., Soulsby, E.J.L. (Eds.), 1984. WHO Guidelines for Surveillance, Prevention and Control of Echinococcosis/Hydatidosis. World Health Organization, Geneva, 148 pp. Eckert, J., Deplazes, P., Craig, P.S., Gemmell, M.A., Gottstein, B., Heath, D., Jenkins, D.J., Kamiya, M., Lightowlers, M., 2001a. Echinococcosis in animals: clinical aspects, diagnosis and treatment. In: Eckert, J., Gemmell, M.A., Meslin, F.-X., Pawlowski, Z.S. (Eds.), WHO/OIE Manual on Echinococcosis in Humans and Animals: A Public Health Problem of Global Concern. World Organisation for Animal Health and World Health Organization, Paris, pp. 72–99. Eckert, J., Schantz, P.M., Gasser, R.B., Torgerson, P.R., Bessonov, A.S., Movsessian, S.O., Thakur, A., Grimm, F., Nikogossian, M.A., 2001b. Geographic distribution and prevalence. In: Eckert, J., Gemmell, M.A., Meslin, F.-X., Pawlowski, Z.S. (Eds.), WHO/OIE Manual on Echinococcosis in Humans and Animals: A Public Health Problem of Global Concern. World Organisation for Animal Health and World Health Organization, Paris, pp. 100–142. Huldt, G., Johansson, S.G.O., Lantto, S., 1973. Echinococcosis in northern Scandinavia. Arch. Environ. Health 26, 36–40. Kojola, I., 2000. Suurpetojen lukumäärä ja lisääntyminen vuonna 1999. Riistantutkimuksen Tiedote, vol. 165. Finnish Game and Fisheries Research Institute, Helsinki, pp. 1–8. Kojola, I., 2001. Suurpetojen lukumäärä ja lisääntyminen vuonna 2000. Riistantutkimuksen Tiedote, vol. 175. Finnish Game and Fisheries Research Institute, Helsinki, pp. 1–6. Kummeneje, K., 1982. Bekjempelse av ekinokokkose (hydatidose) hos rein og hund. Status slaktesesongen 1981/82 ved Kautokeino Slakteri. Norsk Veterinaertidsskrift 94, 6. Lindholm, Å., Lantto, S., 1968. Hydatid disease in Northern Sweden. Arch. Environ. Health 17, 685–688. Maijala, R., Haukisalmi, V., Henttonen, H., Hirvelä-Koski, V., Kauhala, K., Kilpelä, S.-S., Meri, T., Nylund, M., Tenhu, H., Saari, S., 2001. Risk Assessment of the Spread of Echinococcus multilocularis into and within Finland. National Veterinary and Food Research Institute (EELA), Helsinki. Maijala, R., Haukisalmi, V., Henttonen, H., Hirvelä-Koski, V., Kauhala, K., Kilpelä, S.-S., Lavikainen, A., Oksanen, A., Tenhu, H., Vahteristo, L., 2002. Risk Assessment on Echinococcus granulosus in Finland. National Veterinary and Food Research Institute (EELA), Helsinki. Myrseth, O., 1956. Echinokokksykdommen i Finnmark. Tidskr. Norske laegefor. 76, 867–871. Oksanen, A., Laaksonen, S., 1995. Ekinokokin jälkilypsy: kuusamon koirien suuri ulostetutkimus talvella 1993. Suom. Eläinlääkär. 101, 168–172. Pawlowski, Z.S., Eckert, J., Vuitton, D.A., Ammann, R.W., Kern, P., Craig, P.S., Dar, K.F., De rosa, F., Filice, C., Gottstein, B., Grimm, F., Macpherson, C.N.L., Sato, N., Todorov, T., Uchino, J., von sinner, W., Wen, H., 2001. Echinococcosis in humans: clinical aspects, diagnosis and treatment. In: Eckert, J., Gemmell, M.A., Meslin, F.-X., Pawlowski, Z.S. (Eds.), WHO/OIE Manual on Echinococcosis in Humans and Animals: A Public Health Problem of Global Concern. World Organisation for Animal Health and World Health Organization, Paris, pp. 20–71. Pöysti, H., Pöysti, E., 1969. Havaintoja ekinokokkoosista (E. granulosus) poroissa (observations on echinococcosis (E. granulosus) in reindeer). Eläinlääkärilehti 75, 135–137. Rausch, R., 1956. Studies on the helminth fauna of Alaska. The occurrence of Echinococcus multilocularis Leuckart, 1863, on the mainland of Alaska. Am. J. Trop. Med. Hyg. 5, 1086–1092. Rausch, R.L., 1995. Life cycle patterns and geographic distribution of Echinococcus species. In: Thompson, R.C.A., Lymbery, A.J. (Eds.), Echinococcus and Hydatid Disease. CAB International, Wallingford, pp. 89–134. Rein, K., 1957. Echinokokksykdommen i Nord-Norge. Nord. Med. 28, 1853–1857. Roneus, O., 1974. Prevalence of echinococcosis in reindeer (Rangifer tarandus) in Sweden. Acta Vet. Scand. 15, 170–178. Skjenneberg, S., 1959. Ekinokokkose hos rein i Kautokeino. Nord. Vet. Med. 11, 110–123.
192
V. Hirvelä-Koski et al. / Veterinary Parasitology 111 (2003) 175–192
Sweatman, G.K., Williams, R.J., 1963. Comparative studies on the biology and morphology of Echinococcus granulosus from domestic livestock, moose and reindeer. Parasitology 53, 339–390. Söderhjelm, L., 1946. Echinococcus Hydatidosus Hos Rein (Rangifer tarandus). Skand. Vet. Tidskr. 36, 378–381. Thomas, D.C., 1996. Prevalence of Echinococcus granulosus and Taenia hydatigena in caribou in north-central Canada. Rangifer (special issue no. 9), 331–336. Thompson, R.C.A., 1995. Biology and systematics of Echinococcus. In: Thompson, R.C.A., Lymbery, A.J. (Eds.), Echinococcus and Hydatic Disease. CAB International, Wallingford, pp. 1–50. Wilson, J.F., Diddams, A.C., Rausch, R.L., 1968. Cystic hydatid disease in Alaska. Am. Rev. Resp. Dis. 98, 1–15.