Rapid communications Detection of a high-endemic focus of Echinococcus multilocularis

The Danish surveillance programme for Echinococcus multilocularis was initiated in September 2011, and so far 679 wild carnivores have been examined. In April 2012, one infected fox was detected in Højer near the Danish-German border, and in January 2013 three additional foxes from the same area were found infected. Local prevalence in the area was 31% (four of 13 foxes) which is a new epidemiological situation calling for reevaluation of the national risk management.

The Danish surveillance programme for Echinococcus multilocularis was initiated in September 2011, and so far 679 wild carnivores have been examined.In April 2012, one infected fox was detected in Højer near the Danish-German border, and in January 2013 three additional foxes from the same area were found infected.Local prevalence in the area was 31% (four of 13 foxes) which is a new epidemiological situation calling for reevaluation of the national risk management.
As part of the surveillance for the fox tapeworm Echinococcus multilocularis, one red fox (Vulpes vulpes) was found positive in April 2012.The animal had been shot in November 2011 in the Højer area, Jutland in the southern part of Denmark, less than 10 km north of the border between Denmark and Germany [1].In January 2013, E. multilocularis was detected in a further three foxes, one had been shot in September 2012, the other two in November 2012.A total of 13 foxes from this location have been examined corresponding to a local prevalence of 31% (95% confidence interval (CI): 7-55) in foxes.These were the first findings of E. multilocularis in mainland Denmark.

Background
E. multilocularis is endemic in large parts of Europe, and has been detected with increasing prevalence and geographical spread during the last decades, including in countries bordering Denmark, such as Germany [2], the Baltic States [3], and most recently Sweden [4].Humans may be accidental intermediate hosts and develop alveolar echinococcosis , one of the most severe zoonotic infections in the northern hemisphere.Infections in humans are rare but cause considerable public health concern due to treatment costs and high mortality if left untreated [5].In Denmark, infection with E. multilocularis is notifiable in all animal species, but not for human cases.E. multilocularis was discovered for the first time in Denmark in 2000 in three of 1,040 foxes (0.3%).However, all infected foxes were from the Copenhagen area (Zealand) which corresponds to a local prevalence of 0.9% (three of 340 foxes) [6].No additional national surveillance has been conducted in wild carnivores, but one of 169 clinical samples from domestic Danish cats, submitted to a diagnostic German laboratory for routine analyses in 2004-05, was found positive [7].

Surveillance of Echinococcus multilocularis in wild carnivores, 2011-13
Following the detection of E. multilocularis in a Swedish fox in 2011 [4], a surveillance programme for Echinococcus in wild carnivores was initiated in Denmark in September 2011.Included in the surveillance were road-killed and hunted animals collected by the Danish Nature Agency or by voluntary hunters.To date, a total of 679 wild carnivores, namely 546 foxes, 129 racoon dogs (Nyctereutes procyonoides), three badgers (Meles meles) and one wolf (Canis lupus), have been examined by the sedimentation and counting technique [8].The geographical origin of the tested foxes and raccoon dogs is shown in Figure 1.
The first positive fox was shot in November 2011 in Jutland close to the village Højer, 8 km north of the border to Germany (Figure 1), and tested positive in April 2012.The fox was an adult male harbouring 20 adult tapeworms.In addition to morphological identification, these worms were analysed by PCR amplification and sequencing of the 12S rRNA gene [9], revealing a 200 bp product that was 100% identical to E. multilocularis sequences in GenBank, e.g.JX068642.Subgenotyping by fragment size analysis of the EmsB microsatellite marker [10] was done to compare the genetic profile with other European isolates (Figure 2).The results revealed no close relationship to other isolates analysed so far.
In January 2013, three additional foxes from the same area, shot within a radius of 10 km, were detected positive for E. multilocularis by the sedimentation and counting technique.These foxes, all adult females, were shot in September and November 2012, and harboured two, seven and 27 adult tapeworms, respectively.Molecular analysis of these worms is ongoing.Until now a total of 13 foxes (of which four were positive) and three racoon dogs (all negative) from this area have been examined for infection with E. multilocularis corresponding to a local prevalence of 31% (95% confidence interval (CI): 7-55) in foxes.Based on the preliminary surveillance data, the countrywide prevalence of E. multilocularis is 0.7%.So far infection with E. multilocularis has not been detected in wild carnivores other than foxes in Denmark.

Discussion
The Danish E. multilocularis isolate did not cluster closely with any other European isolates of fox origin sub-genotyped until now.Hence, introduction from neighbouring countries cannot be documented on the present basis.
E. multilocularis was first detected in Denmark more than a decade ago [6], but has never before been detected outside Zealand.However, as no surveillance was in place, the parasite may not have been detected.The temperate climate of Denmark allows the survival of E. multilocularis eggs for extended periods, and rodents, implicated as intermediate hosts of the parasite in other European countries, are prevalent [11].Thus, conditions for the establishment and spread of infection are present, although alveolar echinococcosis in intermediate and aberrant hosts has not yet been detected, and there is no information on any autochthonous human cases.
The samples collected for the present study were representative of the whole country.Nevertheless, the current prevalence of E. multilocularis in Danish wildlife is based on analyses of a relatively low number of samples and it is therefore too early to conclude whether there is a general increase in the prevalence on a national level.Even in low-endemic regions, local foci with high prevalence of E. multilocularis in foxes are known [12,13].A consistently high prevalence of 35-65% of E. multilocularis has been registered in foxes in endemic European countries, where human cases appear, and foxes are believed to be responsible for most of the environmental contamination with E. multilocularis eggs [14].Thus, a local prevalence of over 30% in foxes is worrying, and even if based on a small number of foxes, poses an increased risk of transmission to humans as well as dogs and cats. in an increased incidence of alveolar echinococcosis among humans [8].However, as alveolar echinococcosis spreads in a tumour-like manner it can be misdiagnosed as liver cancer.Alveolar echinococcosis should therefore be considered as a differential diagnosis, and may become increasingly important in the future.
Worm burdens detected in Danish foxes so far have been low, which is a diagnostic challenge.On the other hand, fewer worms excrete fewer eggs and these foxes probably do not contaminate the environment as much as foxes with large worm burdens.Nevertheless, a local prevalence of this magnitude emphasised the need for re-evaluating risk management and risk communication in the region, and calls for increased awareness among veterinarians as well as physicians.

Figure 1
Figure 1Geographical distribution of all foxes and raccoon dogs analysed as part of the Danish Echinococcus multilocularis monitoring programme, September 2011-January 2013 (n=675)