Mitochondrial markers
DNA sequences/fragments for the Cox1 derived gene marker (785 bp) could be obtained in 472 of the 505 specimens (93.5%). For 85 of 101 foxes (84.2%), the Cox1 marker was sequenced for all five worm isolates obtained from each fox. Four different single-nucleotide polymorphisms (SNP) were detected in the sequence alignment of the 472 samples (Additional file 1: Figure S2) for an average of 785 bp sequenced of the 1608 bp of the complete Cox1 gene (48.8%).
Cox1
Phylogenetic analysis of the Cox1 gene part sequences, including all the Echinococcus species, assigned all samples examined here to the species E. multilocularis (Additional file 1: Figure S3). These samples form a separate clade within the monophylum E. multilocularis. The Cox1 data also confirm the monophyletic group that includes E. ortleppi (G5) and the genotypes G8, G7, and G6 belonging to the E. canadensis cluster, with high bootstrap values. E. oligarthra is a sister group of this clade in this analysis, but with low bootstrap support. Echinococcus equinus (G4) and E. granulosus sensu stricto (s.s.) (G1 and G3) also form a clade together. Both E. shiquicus and E. vogeli are assigned to independent branches.
One SNP in a Cox1 gene fragment was detected in a single specimen obtained from fox Fu/2009/1607 and in all five worm specimens from fox Fu/2011/1869 at position 9528 (G to T) as compared to the reference sequence AB018440 (obtained from an alveolar lesion isolated from a naturally infected vole [Clethrionomys rufocanus] in Hokkaido, Japan) (https://www.ncbi.nlm.nih.gov/nucleotide/) [53]. One E. multilocularis specimen isolated from fox Fu/2011/420 showed a different SNP at nucleotide position 10146 (C to T). The remaining four parasite isolates did not differ from the other isolates from Brandenburg and North Rhine-Westphalia with regard to the Cox1 marker sequence. At nucleotide position 9625, a further SNP was detected in two parasite isolates of fox Fu/2012/1527 (C to T). Unfortunately, not all five worm isolates obtained from this fox could be sequenced. One out of five E. multilocularis specimens isolated from fox Fu/2011/564, showed a SNP at position 9638 (A to G).
Three of the four SNPs resulted in an amino acid exchange. Leucine was replaced by phenylalanine, glycine by cysteine, and alanine by valine.
Nad1
The sequence of the Nad1 gene fragment was determined for 470 specimens (93.1%). This marker was sequenced in all five worm isolates from 84 foxes (83.2%). Only two different SNPs were detected in the Nad1 gene (Additional file 1: Figure S4) in an average of 379 bp sequenced of 894 bp of the complete Nad1 gene (42.4%).
Based on the phylogenetic analysis performed with the Nad1 sequences, the isolates of the present study could also be assigned to the species E. multilocularis (Additional file 1: Figure S5). Our samples again form a separate clade within the monophylum E. multilocularis. Here, the E. multilocularis clade is a sister group of E. shiquicus. All clades were confirmed with high bootstrap values. The same is true for the monophyletic group formed by E. ortleppi (G5) and the G8, G7, and G6 genotypes from the E. canadensis cluster. Echinococcus granulosus s.s. (G1 and 3) and E. equinus (G4) were each assigned to an independent branch between these clades. The bootstrap ratios are lower than in the clades described above. Both E. oligarthra and E. vogeli form independent branches in this analysis.
Three of the five worm specimens isolated from fox Fu/2009/2374 displayed a SNP in Nad1 at position 7911 (G to A). All five parasite specimens obtained from fox Fu/2009/1860 showed a SNP at nucleotide position 8030 (A to G). One of the two SNPs resulted in an amino acid exchange (glycine to serine).
ATP6
Sequences for ATP6 could be determined in 479 samples (94.9%). The marker was sequenced in all five worm isolates recovered from 88 foxes (87.1%). Four different SNPs were detected in the sequence alignment of the ATP6 gene (Additional file 1: Figure S6) in an average of 516 bp, i.e. the complete ATP6 gene (100%).
Phylogenetic analysis revealed that all ATP6 gene sequences determined in this study could be assigned to the species E. multilocularis (Additional file 1: Figure S7). Here, E. vogeli forms a clade with E. multilocularis, but with little bootstrap support. The monophyletic group, which includes E. ortleppi and the genotypes G8, G7, and G6 from the E. canadensis cluster, could be confirmed with high bootstrap values. Echinococcus oligarthra is a sister group of this clade, but with low bootstrap support. Echinococcus equinus and E. granulosus s.s. together also form a clade. Echinococcus shiquicus was assigned to an independent branch.
In three foxes (Fu/2011/1533, Fu/2011/1551, and Fu/2009/1042), a SNP was detected at the same nucleotide position (6147) in at least one of the five worm sequences (C to T). Four of five parasite sequences of fox Fu/2011/1551 showed a change at this nucleotide position. In the cases of foxes Fu/2011/1533 and Fu/2009/1042, this SNP was only detected in one of the five parasite specimens recovered from these animals. In all five E. multilocularis specimens recovered from fox Fu/2012/1590, a SNP was detected at nucleotide position 5934 (T to C), which was not found in any parasite specimen of any other fox. At position 6247, the ATP6 sequences of all five parasite isolates obtained from fox Fu/2009/1828 differed from the reference sequence and from all other sequences (C to T). The ATP6 sequences of all five worm specimens recovered from fox Fu/2009/1860 exhibited a SNP at position 6375 (C to T).
Three of the four SNPs resulted in amino acid changes (alanine to valine, serine to proline, and histidine to tyrosine).
To increase the robustness of previous phylogenetic analyses, the sequence data were concatenated and re-analyzed for a total of 3189 aligned bp. All concatenated sequences determined in this study could be assigned to the species E. multilocularis (Additional file 1: Figure S8).
Also with the concatenated dataset, our samples form a separate clade within the monophylum E. multilocularis. Here, the E. multilocularis clade is a sister group of E. shiquicus. Both E. oligarthra and E. vogeli form independent branches in this analysis. Within the E. granulosus s.l. group, E. ortleppi and E. canadensis genotypes (G6-G8) clustered together. Echinococcus granulosus s.s. (G1) and E. equinus (G4) are located on separate branches within this group. All clades were confirmed with high bootstrap values (Additional file 1: Figure S8).
In conclusion, infections with mixed genotypes as determined by the Cox1 marker were detected in four foxes, and another one using the Nad1 marker in another fox. Three foxes showed multiple infections with E. multilocularis genotypes, which differed with regard to the ATP6 marker.
EmsB microsatellite analysis and comparison with mitochondrial genotyping
From the total of 505 E. multilocularis specimens, EmsB microsatellite profiles could be determined for 490 (97.0%), and EmsB profiles were obtained for all five worm specimens isolated from each fox for 91 out of 101 foxes (90.1%). For 15 (3.0%) specimens, definitive visual determination of the profile was not possible.
We detected four different profiles (D, E, G, and H) and some parasites that could not be unambiguously assigned to an existing profile (designated as K), but were further analyzed as described in the next section. Profile E could only be detected in a single E. multilocularis isolate from North Rhine-Westphalia. In 80 of 101 foxes (79.2%), genotyping information for the mitochondrial markers (Cox1, Nad1, and ATP6) and the EmsB profiles could be determined for all five worm isolates obtained from each fox. Profile D was found in 194 (38.4%), G in 257 (50.9%), H in 38 (7.5%), and E in one (0.2%) of the 505 worm specimens. The differences in the proportions of the profiles were statistically significant (Fisher’s exact test; P-value = 0.0041).
In 14 foxes (10 from Brandenburg and four from North Rhine-Westphalia) the EmsB profile of at least one E. multilocularis specimen differed from the profiles of the remaining four specimens from the respective foxes.
For the foxes Fu/2011/564, Fu/2009/1607, Fu/2011/420, and Fu/2012/1527, in which one worm isolate differed from the remaining four worm isolates by a SNP in the Cox1 gene, it was shown that the EmsB profile of the respective specimen also differed from the other four specimens obtained from the same fox. Thus, all four E. multilocularis specimens from fox Fu/2011/564 belonged to the EmsB profile H, and the specimen Fu/2011/564-5 had the profile D. The profile G was found four times in fox Fu/2009/1607 and profile H once in Fu/2009/1607-3. The SNP in the Cox1 gene of the worm isolate 3 was also detected in all five parasite specimens isolated from fox Fu/2011/1869. All isolates from this fox belonged to the EmsB profile H. In fox Fu/2011/420, profile D was found in four specimens and profile H in one parasite. In fox Fu/2012/1527, profile D was found four times and profile G once. No agreement was found in EmsB profile heterogeneity with the SNPs in the remaining two mitochondrial markers Nad1 and ATP6.
Validation of the visual determination of EmsB profiles
To evaluate the reliability of the visual determination of EmsB profiles and to determine their distinctiveness, supervised self-organizing KN analysis was performed and the results displayed in SOM. The standardized numerical EmsB profiles found in this study were used as the data basis. Due to a change in the standard for capillary gel electrophoresis, 38 samples had to be removed from the data set, so that 427 samples remained for KN analysis. Profiles D and E were combined into one group, because only one of the 490 samples for which an EmsB profile could be determined was assigned to profile E. After the number of expected groups (equivalent to the number of expected EmsB profiles) had been set, classification by the KN was carried out using the “xyf” function in R package “kohonen.” In the graphical visualization of the results, circles represent the groups of profiles. Circles with the same color belong to the same group. The visually determined profiles are shown in different colored circle sectors and were assigned to the groups using the “xyf” function.
The analysis revealed four groups predicted by the KN according to the EmsB profiles, which are identified by four different colors (Fig. 2). Each EmsB profile corresponded perfectly to the assumed group. The samples that could not be visually assigned to any profile formed a separate group in this analysis (profile K).
To examine whether the samples that could not be visually assigned to any profile could be grouped with any of the established profiles D, G, or H, the existence of only three groups was assumed and the analysis repeated. Under these conditions, the visually determined profile G and the samples that could not be clearly assigned to any profile (K) formed one group (Fig. 3). Furthermore, a small part of the samples that could not be allocated to any profile (K) clustered with profile H. None of the samples that could not be visually attributed to any profile was allocated to profile D.
Unsupervised KSN analysis mapped the EmsB data into four groups/clusters, confirming the correctness of the visually classified EmsB profiles (D (red nodes), G (green nodes), H (light blue nodes), and unknown profiles designated as “K” (dark blue node) (Fig. 4a, b).
The validity of the visual profile determination was further examined by Sammon’s nonlinear mapping with k-means clustering. When the existence of three clusters was assumed, the resulting profile groups corresponded to the visually determined profiles (Fig. 5). These groups were spatially separated from each other. The samples which could not be visually assigned to any profile scattered around the cloud representing samples with profile G.
Hierarchical clustering analysis also classified the EmsB genotyping data of the E. multilocularis isolates into four groups when we applied a threshold of 0.08 for the genetic distance [18] (Fig. 6). The profiles D, G, and H were clustered in three separate clusters, with the fourth cluster consisting mainly of samples representing patterns that could not be assigned to any known EmsB profile and single isolates representing EmsB profiles D or G.
These results confirm in multiple ways the validity of the visual determination of profiles. It seems likely that samples that could not be attributed to any established profile belong to at least one separate profile.
Spatial distribution of genotypes
Due to the limited number of SNPs in the sequences of the mitochondrial markers Cox1, Nad1, and ATP6, it was not possible to draw any conclusions regarding the spatial distribution of E. multilocularis on the level of the federal state of Brandenburg or in the study area in North Rhine-Westphalia (Additional file 1: Figure S9).
When the spatial coordinates of E. multilocularis-infected foxes (n = 90) with identical EmsB profiles for all sampled parasite specimens were plotted on a map, it became evident that profile G was predominantly detected in northwestern Brandenburg in the districts of Prignitz and Ostprignitz-Ruppin (Fig. 7). In contrast, profile D was predominantly found in central and southern Brandenburg. Profile H was found in three of the foxes, two of which came from Brandenburg, and the third one from North Rhine-Westphalia. Profile D was found in two foxes and profile G in a single fox in North Rhine-Westphalia.
Spatial analysis by searching for high rate clusters with a Bernoulli model using SaTScan revealed two clusters (Fig. 7). Cluster 1 is located in northwestern Brandenburg and comprises foxes with the E. multilocularis specimens of profile G. Cluster 2 is formed by parasites of the profile D. The geographical center of this profile is south of Berlin in the middle of Brandenburg and extends to the central and southern parts of the federal state. No separate cluster was identified for profile H and the foxes (n = 2) assigned to it, nor could this profile be included in one of the two confirmed clusters.