Echinococcus multilocularis confirmed in Romania...Sci Parasitol 11(2):89-96, June 2010 ISSN 1582-1366 ORIGINAL RESEARCH ARTICLE 89 Echinococcus multilocularis confirmed in Romania

Sci Parasitol 11(2):89-96, June 2010 ISSN 1582-1366 ORIGINAL RESEARCH ARTICLE

89

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

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

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-Austria-Germany to Poland and Slovak Republic and then to Ukraine and Hungary (figure 1) is


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feasible or not. The EURECHINOREG data (2003) show the same way of the E.

multilocularis extension (Sikó Barabási and Cozma, 2008).

Figure 1. The suspected direction of the E. multilocularis

propagation route (Sikó Barabási and Cozma, 2008)

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). 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. 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.

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

Country Year Host Reference

Poland 2001-2004

fox Malczewski et al. (2008)

Slovenia 2006-2007

man Logar et al. (2007)

1988 man Sréter et al. (2003) Hungary 2003 fox Sréter et al. (2003; 2004) 1951 man Altintas (2003) Bulgaria 1980 rodent Genov et al. (1980)

Greece 1998 man Kolárová (1999) 1934 man Uysal and Pakson (1986) 1939 man Altintas (2003)

Turkey

1965 fox Altintas (2003) Moldavia 1961 rodent Bessonov (2000; 2002) Ukraine 1957,

2006, 2008

fox Bessonov (2000; 2002); Kharcenko et al. (2008); Varodi et al. (2006)

Armenia 1958 man Taira et al (2003) 1957, 1958

rodent Bessonov (1998; 2002) Byelorussia

2001, 2003

fox Shimalov and Shimalov (2001; 2003)

2003 muskrat Mažeika et al. (2003; 2009)

2003 dog, pig Bružinskaitė et al. (2007)

Lithuania

2007 fox, man

Bružinskaitė et al. (2007)

Latvia 2008 fox Bagrade et al. (2008) Estonia 2005 fox Moks et al. (2005)

1957 fox Bessonov (2002) 1966 wild cat Taira et al (2003)

Russia

1970, 1972, 1998

fox, man

Bessonov (1998); Lukashenko et al. (1970); Lukashenko (1971; 1972)

Materials and methods

Sampling and examination of red foxes

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).


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Figure 2. Metacestode structures containing protoscoleces in Arvicola terrestris (from Sikó Barabási et

al., 1995)

Table 2. Previous investigations for the presence of cestodes of genus Echinococcus in dogs and foxes from

Romania

Host

(n)

Prevalence

(%)

Examination

technique Reference

Fox (50)

- Necropsy Gherman et al. (2002)

Fox (33)

5.12 Necropsy Sikó Barabási and Sándor (2007)

Fox (197)

2.03 Necropsy Sikó Barabási et al. (2008)

Fox (25)

18.00 CoproELISA Seres and Cozma (2008)

Fox (23)

30.00 CoproELISA Seres et al. (2008b)

Dogs (40)

12.50 CoproELISA Seres et al. (2006)

Dogs (77)

28.60 CoproELISA Seres et al. (2007)

Dogs (135)

28.00 CoproELISA Seres et al. (2008a)

The isolated worms were identified and counted. The determined worms were re-counted, 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 CHEKIT-Echinotest biphasic enzyme immunoassay kit, manufactured by IDEXX Laboratories, Netherlands. Statistical analysis

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


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Table 3. Parasitic infestation of foxes small intestines

Class Species Prevalence

(%)

Trematoda Alaria alata 14.97 Dipylidium caninum 9.98 Mesocestoides lineatus 40.10 Taenia pisiformis 22.00 Taenia hydatigena 6.00 Taenia multiceps 4.62 Taenia serialis 0.90

Cestoda

Taenia taeniaeformis 2.61 Trichuris vulpis 27.21 Ancylostoma caninum 18.19 Uncinaria stenocephala 15.00

Nematoda

Toxocara canis 29.40

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. 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. Prevalence of E. multilocularis infection

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 tape-like 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.

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

No. County n Infected Prevalence (%)

95% CI

1. AB 23 - 0.0 (0.0-12.2) 2. AR 19 2 10.5 (1.3-33.1) 3. BH 41 6 14.6 (5.6-29.2) 4. BN 53 3 5.7 (1.2-15.7) 5. BV 24 - 0.0 (0.0-11.7) 6. CJ 36 1 2.8 (0.1-14.5) 7. CV 118 2 1.7 (0,2-6.0) 8. HR 41 1 2.4 (0.1-12.9) 9. HD 15 - 0.0 (0.0-18.1)

10. MM 37 4 10.8 (3.0-25.4) 11. MS 36 - 0.0 (0.0-8.0) 12. SM 63 8 12.7 (5.6-23.5) 13. SJ 18 - 0.0 (0.0-15.3) 14. SB 18 - 0.0 (0.0-15.3) 15. TM 19 - 0.0 (0.0-14.6)

TOTAL 561 27 4.8 (3.2-6.9)

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

The length of terminal proglotids was 0.7-1.2 mm. The terminal proglotids represented 25-44% 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).


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The Polymerase Chain Reaction

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

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).

Figure 4. Different morphological aspects of E. multilocularis

The used Primers: UnivSr: 5’-GCGGTGTGTACMTGAGCTAAAC-3’

TaeSf: 5’-YGAYTCTTTTTAGGGGAAGGTGTG-3’

EgS2f: 5’-GTTTTTGTGTGTTACATTAATAAGGGTG-3’

EmN2f: 5’-TGCTGATTTGTTAAAGTTAGTGATC-3’

EmNr: 5’-CATAAATCAATGGAAACAACAACAAG-3’

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

Taenia)

Results Sample

E. granulosus E. multilocularis Taenia

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

1 2 3 4 6 5 7

Figure 5. The sequence analysis

The coproantigen ELISA

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).

Discussions 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.


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Table 6. Correlation between the morphological prevalence, coproantigen ELISA test and PCR

Necropsy Coproantigen

ELISA County No. examined

n % 95 CI n Positive

(Dubious)

PCR

AB 23 0 0.0 (0.0-12.2) 6 0 - AR 19 2 10.5 (1.3-33.1) 16 3 - BH 41 6 14.6 (5.6-29.2) 24 5 + BN 53 3 5.7 (1.2-15.7) 8 (3) - BV 24 0 0.0 (0.0-11.7) 6 0 - CJ 36 1 2.8 (0.1-14.5) 8 (2) - CV 118 2 1.7 (0,2-6.0) 16 2 (4) + HR 41 1 2.4 (0.1-12.9) 16 1 (5) - HD 15 0 (0.0-18.1) 6 0 - MM 37 4 10.8 (3.0-25.4) 22 4 (2) - MS 36 0 (0.0-8.0) 6 0 - SM 63 8 12.7 (5.6-23.5) 25 5 (1) + SJ 18 0 (0.0-15.3) 6 0 - SB 18 0 (0.0-15.3) 6 0 - TM 19 0 (0.0-14.6) 8 (1) -

Total 561 27 4,8 (3.2-6.9) 176 20 (17) 3

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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Echinococcus multilocularis confirmed in Romania...Sci Parasitol 11(2):89-96, June 2010 ISSN 1582-1366 ORIGINAL RESEARCH ARTICLE 89 Echinococcus multilocularis confirmed in Romania