Investigation of Echinococcus multilocularis in Environmental Definitive Host Feces in the Asian and the European Parts of Turkey

A study was carried out to investigate the presence of Echinococcus multilocularis in red foxes (Vulpes vulpes) in two regions of Turkey—central Anatolia (in Asia Minor) and Thrace (in the European part of Turkey). A total of 405 putative fox feces were collected from central Anatolia (186 specimens in 59 locations) and from Thrace (219 specimens in 114 locations). All samples were examined by the flotation and sieving method for taeniid eggs, and positive and putative samples were further analyzed by multiplex PCR. In seven samples from three locations in central Anatolia (5.1%) and in one (0.9%) from Thrace, E. multilocularis DNA was amplified, and this result was confirmed with another PCR specific for E. multilocularis. In addition, Echinococcus granulosus s.l. was found in two (0.5%) of the samples. Although alveolar echinococcosis (AE) is known as a serious zoonosis in Turkey, this is the first field study detecting E. multilocularis in collected fecal samples documenting the environmental contamination with eggs of this zoonotic parasite.

The aim of this study is to demonstrate the presence of E. multilocularis in field samples in two regions of Turkey: Central Anatolia where AE in humans is known to occur (7) and Thrace, where there is only historical evidence of possible endemicity.

MaTerials anD MeThODs study area
Turkey is located on 36°-42° North and 26°-45° East longitude and consists of two parts-Anatolia (Asia Minor) in Asia and Thrace in Europe. The study was performed in five cities-two (Kayseri and Nevşehir) in central Anatolia and three (Kırklareli, Edirne, and Tekirdağ) in Thrace (Figure 1). There are no records on the fox population in Turkey. Consequently, fox feces were collected randomly from different locations, each at least 1 km distance from the other. All locations were situated in rural areas, including villages and fields with some locations around fox holes.

Diagnosis of E. multilocularis
All fecal samples were stored at −80°C for at least a week before analysis as safety precautions. Diagnosis of E. multilocularis consisted of three steps; determination of the presence of taeniid eggs in the samples; isolation of taeniid eggs; and molecular analysis of them.
All fecal samples were examined and taeniid eggs were concentrated by the flotation and sieving method, modified by Mathis et al. (8). Samples with identifiable taeniid eggs or with particles with similar size and shape as taeniid eggs and six negative samples collected in Thrace, where an E. multilocularis positive fox was identified in the 50 years previously (5), were further investigated by PCR. DNA was extracted by alkaline lysis (9) and amplified using the multiplex-PCR protocol according to Trachsel et al. (10). All positive samples for E. multilocularis were confirmed with the PCR protocol according to Stieger et al. (11).
In samples with Echinococcus spp. DNA amplification, the fox origin was confirmed by multiplex PCR as described by Nakao et al. (12). Positive control DNA extracted by tissue from the fox's tongue and dog's blood. Size of PCR products were 165 bp for fox and 355 bp for dog samples.
resUlTs DNA was extracted from 60 of 405 putative fox fecal samples. E. multilocularis DNA was amplified in eight samples in both areas investigated (Table 1; Figure 1). From Anatolia, seven samples were found to be positive for E. multilocularis. Six of these samples contained other Cestoda spp. DNA. In one sample, E. multilocularis, Echinococcus granulosus, and non-Echinococcus spp. cestode DNA was amplified.
In Thrace, E. multilocularis DNA was amplified in one of six egg negative samples. Furthermore, E. granulosus DNA was identified in one sample, and 25 samples contained DNA of non-Echinococcus cestodes.
All samples with Echinococcus spp. DNA amplification were identified by PCR as being of fox origin.

DiscUssiOn
Turkey is a highly endemic region for E. multilocularis. Since 1939, when first reported, more than 750 human cases of AE  (5). Interestingly, further north, E. multilocularis has been described in rodents in Bulgaria and in foxes in Romania (16). In total, in this study, eight fox feces (1.9%) in four locations (2%) were positive for E. multilocularis. Three locations were from the central Anatolia and one from Thrace. Our finding of a positive fecal sample in Thrace, although negative for taeniid eggs, was confirmed by two PCRs targeting different mitochondrial genes. This therefore confirms that E. multilocularis is still endemic in Thrace in the same area where it was identified in 1960s. Interestingly, however, no cases of AE are known from this region, except possibly three suspect cases (17). By contrast, Anatolia (Asia Minor) is well known to be an endemic area for AE in humans, and recently the first infected fox has been documented (6). Between 1980 and 2010, 13 AE cases were reported from the city of Kayseri and one from the city of Nevşehir (7). The number of AE cases in humans increases gradually in an eastward direction in Turkey, with most cases recorded from East Turkey (17)(18)(19).  (24).
In this preliminary study, we focused on the environmental contamination with E. multilocularis eggs based on fecal samples. However, we cannot exclude that the morphological identification of the samples was 100% specific for fox samples. Poulle et al. documented that the morphological assessment of fecal samples has a sensitivity of around 83% (95% confidence intervals, 61-95%) (25). Therefore, for all Echinococcus positive samples, the fox origin was confirmed by PCR even in the two samples with E. granulosus-specific amplification. Turkey is a well-known endemic area of E. granulosus (2), and E. granulosus prevalence in foxes ranged up to 50% in Australian foxes (26).

aUThOr cOnTribUTiOns
AG and PD developed the study protocol and oversaw all procedures. AG, FG, CB, MA, and ŞU organized to collection and investigation of the materials. AG and PD drafted the manuscript, and the final version was approved by all authors.

FUnDing
Our pilot study was supported by Institute of Parasitology, University of Zurich.