Echinococcus multilocularis confirmed in Romania - Zooparaz.net

Sci Parasitol 11(2):89-96, June 2010

ORIGINAL RESEARCH ARTICLE

ISSN 1582-1366

Echinococcus multilocularis confirmed in Romania Sándor Sikó Barabási1, Peter Deplazes2, Vasile Cozma3, Simona Pop4, Csaba Tivadar5, Ioan Bogolin6, Raluca Popescu7

1 - Echino-News Assoc. Romania, 520036 Sf. Gheorghe, Ciucului str., 149, Covasna county, Romania. 2 - University of Zürich, Institute of Parasitology, Winterthurerstr. 266a, 8057 Zürich, Switzerland. 3 - University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, Department of Parasitology and Parasitic Diseases, Calea Mănăştur 3-5, 400372 Cluj-Napoca, Romania. 4 - Veterinary and Food-Safety Laboratory Bihor county, Romania. 5 - Veterinary and Food-Safety Laboratory Satu Mare county, Romania. 6 - Veterinary and Food-Safety Direction Bistriţa-Năsăud county, Romania. 7 - Veterinary and Food-Safety Laboratory Covasna county, Romania. Correspondence: [email protected]

Abstract. The occurrence of the zoonotic tapeworm, Echinococcus multilocularis in definitive hosts, in intermediate or aberrant hosts, has been described in many east-European countries. So far, the adult stage of the parasite was not reported in red foxes from Romania. Therefore, between August 2007 and March 2010, we have investigated 561 European red foxes (Vulpes vulpes L.) for intestinal helminths. The small intestines of the investigated foxes were infected in 14.97% with trematodes, 91.08% with cestodes and 91.44% with nematodes. In 27 (4.81%) of the samples we have detected E. multilocularis (between 8 and >100 individuals per fox). The morphological identification was confirmed by a multiplex PCR, followed by sequence analysis, and by coproantigen ELISA CHEKIT test. This is the first confirmation for the presence of E. multilocularis in the Romanian fox population.

Keywords: Echinococcus multilocularis; Vulpes vulpes; Red fox; Intestinal helminths; Romania. Received 17/02/2010. Accepted 28/05/2010.

Introduction

suggested by many authors (Kolárová, 1999; Dubinský et al., 2001; Sréter et al., 2004; Letková et al., 2006; Reiterová et al., 2006; Kharcenko et al., 2008) we aimed to check whether the suspected propagation route of the parasite from Switzerland-AustriaGermany to Poland and Slovak Republic and then to Ukraine and Hungary (figure 1) is

The area considered endemic for Echinococcus multilocularis covers most parts of Central and Eastern Europe (Romig et al., 2006). In the last years, the E. multilocularis was reported in red fox (Vulpes vulpes) population from several countries neighboring Romania. Thus, as 89



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Table 1. Reports of E. multilocularis in East European countries

feasible or not. The EURECHINOREG data (2003) show the same way of the E. multilocularis extension (Sikó Barabási and Cozma, 2008).

Country Poland Slovenia Hungary Bulgaria Greece Turkey

Moldavia Ukraine

Armenia Byelorussia

Figure 1. The suspected direction of the E. multilocularis propagation route (Sikó Barabási and Cozma, 2008)

Lithuania

Moreover, the Eastern European reports on E. multilocularis are abundant in these countries (table 1). These aspects put under a question mark the idea of western to eastern spread route of the E. multilocularis, as the parasite was described earlier herein (Sikó Barabási et al., 2009). During studies performed between 1991-1995 in the sub alpine region of the East Carpathian Mountains, E. multilocularis was for the first time diagnosed by morphological identification of larval structures containing protoscoleces in Romania in wild rodents (Arvicola terrestris, Microtus nivalis, M. arvalis, Clethrionomys glareolus) (Sikó Barabási, 1992; 1993; Sikó Barabási et al., 1995) (figure 2).

Year 20012004 20062007 1988 2003 1951 1980 1998 1934 1939 1965 1961 1957, 2006, 2008 1958 1957, 1958 2001, 2003 2003 2003 2007

Latvia Estonia Russia

2008 2005 1957 1966 1970, 1972, 1998

Host fox

Reference Malczewski et al. (2008)

man

Logar et al. (2007)

man fox man rodent man man man fox rodent fox

Sréter et al. (2003) Sréter et al. (2003; 2004) Altintas (2003) Genov et al. (1980) Kolárová (1999) Uysal and Pakson (1986) Altintas (2003) Altintas (2003) Bessonov (2000; 2002) Bessonov (2000; 2002); Kharcenko et al. (2008); Varodi et al. (2006) Taira et al (2003) Bessonov (1998; 2002)

man rodent fox muskrat dog, pig fox, man fox fox fox wild cat fox, man

Shimalov and Shimalov (2001; 2003) Mažeika et al. (2003; 2009) Bružinskaitė et al. (2007) Bružinskaitė et al. (2007) Bagrade et al. (2008) Moks et al. (2005) Bessonov (2002) Taira et al (2003) Bessonov (1998); Lukashenko et al. (1970); Lukashenko (1971; 1972)

Materials and methods Sampling and examination of red foxes

So far, alveolar echinococcosis was found in 2 human cases in Romania (Sikó Barabási, 1992; 1993; Sikó Barabási et al., 1995; Sikó Barabási and Cozma, 2008). Furthermore, larval cestodes with alveolar structures were also reported in sheep and horses. However, the species diagnosis of these findings was not confirmed (Sikó Barabási and Cozma, 2008). Several data collection for the finding of adult stages of E. multilocularis were performed in Romania in dogs and foxes (table 2), but no molecular confirmation was achieved.

Between August 2007 and March 2010, a number of 561 carcasses of red foxes were collected from 15 Transylvanian counties. Most of them were hunted (yearly, between October and March) and some were road kills. Carcasses were transported and stored in individual plastic bags at 4°C. The approximate delay between death and necropsy was 2 days. The small intestine samples were examined by mucus scraping and accounting methods, and intestinal sedimentation method. After several repeated sedimentations, the intestinal content and the scrapped mucus were examined in Petri dishes under a stereomicroscope at a magnification of 66x (as described by Deplazes and Eckert, 1996; Hofer et al., 2000; Duscher et al., 2005).

In this paper, we have systematically investigated foxes from 15 Transylvanian counties for intestinal helminthes especially to investigate the prevalence of E. multilocularis in this region. 90



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The isolated worms were identified and counted. The determined worms were recounted, and subsequently washed and stored in 70% ethanol until the identification. All the storage recipients were labeled. Species identification E. multilocularis was identified according to the overall size of the worm and the shape of uterus in the last mature segment (Taira et al., 2003; Eckert and Deplazes, 2004). To confirm the morphological identification of E. multilocularis a multiplex PCR (Trachsel et al., 2007) followed by sequence analysis was performed with 3 isolates. The methodology is described within results section. Coproantigen ELISA For detecting Echinococcus-specific antigens in 176 fecal samples we have used the CHEKITEchinotest biphasic enzyme immunoassay kit, manufactured by IDEXX Laboratories, Netherlands. Figure 2. Metacestode structures containing protoscoleces in Arvicola terrestris (from Sikó Barabási et al., 1995)

Statistical analysis Prevalence of E. multilocularis (positive/negative) in red foxes were cross tabulated and analyzed using two–tailed Fisher's Exact Test.

Table 2. Previous investigations for the presence of cestodes of genus Echinococcus in dogs and foxes from Romania Host (n) Fox (50) Fox (33)

Prevalence (%) -

Examination technique Necropsy

5.12

Necropsy

Fox (197) Fox (25) Fox (23) Dogs (40) Dogs (77) Dogs (135)

2.03

Necropsy

18.00

CoproELISA

30.00

CoproELISA

12.50

CoproELISA

28.60

CoproELISA

28.00

CoproELISA

Ninety five percent exact binominal confidence intervals (95% CI) for the intensity and prevalence were calculated. Infection intensities for foxes were compared using Mann-Whitney test. All statistical calculations were performed using GraphPad Prism version 4.0 for Windows® (www.graphpad.com).

Reference Gherman et al. (2002) Sikó Barabási and Sándor (2007) Sikó Barabási et al. (2008) Seres and Cozma (2008) Seres et al. (2008b) Seres et al. (2006) Seres et al. (2007) Seres et al. (2008a)

Results Overall helminthic infection The parasitic infestation of the small intestine samples was ploy-specific (table 3). The overall intensity of the infestation was between 1 and over 1000 helminths/fox, with the most prevalent species being Mesocestoides lineatus. 91



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Table 3. Parasitic infestation of foxes small intestines Class

Species

Trematoda Cestoda

Alaria alata Dipylidium caninum Mesocestoides lineatus Taenia pisiformis Taenia hydatigena Taenia multiceps Taenia serialis Taenia taeniaeformis Trichuris vulpis Ancylostoma caninum Uncinaria stenocephala Toxocara canis

Nematoda

Table 4. Number of examined red foxes and prevalence of E. multilocularis

Prevalence (%) 14.97 9.98 40.10 22.00 6.00 4.62 0.90 2.61 27.21 18.19 15.00 29.40

No.

County

1. AB 2. AR 3. BH 4. BN 5. BV 6. CJ 7. CV 8. HR 9. HD 10. MM 11. MS 12. SM 13. SJ 14. SB 15. TM TOTAL

The majority of the cestodes were found in the median thirty part of the jejunum. Nematodes of the Ancylostomatidae family were isolated from the posterior part of the jejunum and the ileum, and Trichuris vulpis was isolated from the cecum.

n

Infected

23 19 41 53 24 36 118 41 15 37 36 63 18 18 19 561

2 6 3 1 2 1 4 8 27

Prevalence (%) 95% CI 0.0 (0.0-12.2) 10.5 (1.3-33.1) 14.6 (5.6-29.2) 5.7 (1.2-15.7) 0.0 (0.0-11.7) 2.8 (0.1-14.5) 1.7 (0,2-6.0) 2.4 (0.1-12.9) 0.0 (0.0-18.1) 10.8 (3.0-25.4) 0.0 (0.0-8.0) 12.7 (5.6-23.5) 0.0 (0.0-15.3) 0.0 (0.0-15.3) 0.0 (0.0-14.6) 4.8 (3.2-6.9)

These aspects demonstrated the existence of some specific ecological locus for the isolated helminths. The length of the cestodes from Taenia and Mesocestoides genera was under 20-25 cm, especially in massif infestations with over1000 helminths/fox. The geographic distribution between the different counties as well as the repartition in correlation with sex and age were not significantly different.

Figure 3. Geographical distribution of E. multilocularis infected foxes (n=561)

Prevalence of E. multilocularis infection The length of terminal proglotids was 0.7-1.2 mm. The terminal proglotids represented 2544% of the total length of the worms. The genital pore position in the gravid segment was anterior to middle of segment. The uterus form was sac-like, without lateral sacculations. The testes number varied between 12 and 24 and they were distributed from the level of the genital pore to posterior end of segment (figure 4). Based on these morphometric parameters the parasites were identified as Echinococcus multilocularis (Sréter et al., 2003).

We have isolated small cestodes from genus Echinococcus from 27 foxes (4.81%). From all the counties investigated, 53.33% had infested foxes (table 4, figure 3). The highest prevalence was in Bihor county (14.63%), in Satu Mare county (12.7%), in Maramureș county (10.81%) and in Arad county (10.52%). Morphology of isolated E. multilocularis The isolated cestodes had an elongated tapelike body with 1.2-2.7 mm in length. They had 3-5 segments. The scolex was armed with two rows of rostellar hooks and four suckers.

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The Polymerase Chain Reaction

From the 27 small intestinal samples infested with E. multilocularis we have isolated 3 samples for a multiplex PCR, followed by sequence analysis. In all 3 isolated samples the presence of E. multilocularis DNA was confirmed (table 5, figure 5).

The taxonomic status of the isolates was also confirmed by a diagnostic polymerase chain reaction (PCR) assay.

Figure 4. Different morphological aspects of E. multilocularis

The used Primers: UnivSr: TaeSf: EgS2f: EmN2f: EmNr:

The coproantigen ELISA

5’-GCGGTGTGTACMTGAGCTAAAC-3’ 5’-YGAYTCTTTTTAGGGGAAGGTGTG-3’ 5’-GTTTTTGTGTGTTACATTAATAAGGGTG-3’ 5’-TGCTGATTTGTTAAAGTTAGTGATC-3’ 5’-CATAAATCAATGGAAACAACAACAAG-3’

The Coproantigen ELISA CHEKIT-Echinotest was positive in 20 samples (11.36%), ambiguous in 17 cases (9.65%) and negative in 149 cases (84.65%). Comparing the results of the morphologically identification prevalence with the coproantigen ELISA test results, we can not establish significant differences. However, the differences observed between CV and HR counties can be explained with the low infestation of the samples collected from these areas (table 6).

Table 5. Multiplex-PCR (adults: E - Echinococcus; T Taenia) Sample

Results E. granulosus

E. multilocularis

Taenia

-

+ + + -

+

E - BH E - SM 1 E - SM 2 T - SM

Discussions 1

2

3

4

5

6

Although, the presence of the E. multilocularis in Romania was suspected, the human and animal intermediate hosts cases were not confirmed by specific diagnostic tests. Furthermore, the adult stages of the parasites isolated from a foxes were identified only by morphological methods. This is the first paper which demonstrates the presence of E.multilocularis in fox populations from Romania.

7

Figure 5. The sequence analysis

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Table 6. Correlation between the morphological prevalence, coproantigen ELISA test and PCR Necropsy County AB AR BH BN BV CJ CV HR HD MM MS SM SJ SB TM Total

No. examined 23 19 41 53 24 36 118 41 15 37 36 63 18 18 19 561

n

% 95 CI

n

0 2 6 3 0 1 2 1 0 4 0 8 0 0 0 27

0.0 (0.0-12.2) 10.5 (1.3-33.1) 14.6 (5.6-29.2) 5.7 (1.2-15.7) 0.0 (0.0-11.7) 2.8 (0.1-14.5) 1.7 (0,2-6.0) 2.4 (0.1-12.9) (0.0-18.1) 10.8 (3.0-25.4) (0.0-8.0) 12.7 (5.6-23.5) (0.0-15.3) (0.0-15.3) (0.0-14.6) 4,8 (3.2-6.9)

6 16 24 8 6 8 16 16 6 22 6 25 6 6 8 176

Coproantigen ELISA Positive (Dubious) 0 3 5 (3) 0 (2) 2 (4) 1 (5) 0 4 (2) 0 5 (1) 0 0 (1) 20 (17)

PCR + + + 3

Bružinskaitė R., Marcinkutė A., Strupas K., Sokolovas V., Deplazes P., Mathis A., Eddi C., Šarkunas M. 2007. Alveolar Echinococcosis, Lithuania. Emerg. Infect. Dis. 13:1618-1619. Deplazes P., Eckert J. 1996. Diagnosis of the Echinococcus multilocularis infection in final hosts. Appl. Parasitol. 37:245-252. Dubinský P., Várady M., Reiterová K., Miterpáková M., Turéeková L. 2001. Prevalence of Echinococcus multilocularis in red foxes in the Slovak Republic. Helminthologia 38:215-219. Duscher G., Prosl H., Joachim A. 2005. Scraping or shaking – a comparison of methods for the quantitative determination of Echinococcus multilocularis in fox intestines. Parasitol. Res. 95:40-42. Eckert J., Deplazes P. 2004. Biological, epidemiological, and clinical aspects of echinococcosis, a zoonosis of increasing concern. Clin. Microbiol. Rev. 17:107-135. Genov T., Svilenov D., Polyakova-Krusteva O. 1980. The natural occurrence of Alveococcus multilocularis in the Microtus nivalis in Bulgaria. C. R. Acad. Bulgare Sci. 33:981-984. Gherman C., Cozma V., Mircean V., Brudaşcă F., Rus N., Detasan A. 2002. Helminthic zoonoses in wild carnivorous species from Romanian fauna. Rev. Sci. Parasitol., 3:17-21. Hofer S., Gloor S., Müller U., Mathis A., Hegglin D., Deplazes P. 2000. High prevalence of Echinococcus multilocularis in urban red foxes (Vulpes vulpes) and voles (Arvicola terrestris) in the city of Zürich, Switzerland. Parasitology 120:135-142.

Conclusion It remains unclear if Romania is a new endemic area and if the parasite was introduced recently or if the parasite was not detected before. Further investigations are in progress.

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