Based on the rarefaction curve, we compared our findings with those of historical studies [ 4 - 6 , 8 , 9 ]. Foxes, for which biological parameters or geographical data were missing, were excluded from analysis. This limited the available dataset for multifactorial analysis to foxes. Correlations between body condition, age, gender and parasite prevalence were determined by ANOVA analysis of variance.
To analyse the presence of E. PCR products were specified by southern blot hybridization, using E.
To confirm the identification of cestode species, a fragment of the mitochondrial cytochrome c oxidase 1 CO1 gene was amplified as described by Bowles et al. PCR amplification of the partial CO1 gene was performed using the following conditions: Sequence PCR was performed under the following conditions: DNA sequences were assembled, edited, and analysed with BioNumerics version 6. Obtained CO1 gene sequences were compared to reference sequences present in Genbank after subtraction of the primer sequences.
Cluster analysis of the sequences was conducted using the unweighted neighbour-joining algorithm of the BioNumerics program. Bootstrap proportions were calculated by the analysis of replicates for neighbour-joining trees. Available CO1 sequences of cestodes and trematodes from Genbank were included in the alignment. Unequivocally identified Alaria alata isolates from foxes from this study served as out-group in phylogenetic analysis. In total, foxes were collected. This age distribution of shot foxes indicates high hunting pressure as found in previous studies [ 30 , 40 ].
Seventeen helminth species were identified from our reference data set of foxes. Analysis of fox parasite species by rarefaction method. Number of co - infections per age group and per gender. Male foxes peak at three to four co-infections, females nine months of age and younger peak at two to three co-infections. Male foxes exhibit the highest numbers of co-infection 8. Zero co-infections mean no infection at all. Total number of foxes is The prevalences of T. Methods used for detection and speciation. Species number 6, 9 and 10 were obtained from heart and lung washings for which 96 foxes were available.
Four out of four urine bladders were found positive for this species, but prevalence was not extrapolated from this limited number of analyses. The prevalence of T. The prevalences of Strongyloides sp. Trichuris vulpis , Angiostrongylus vasorum , Mesocestoides litteratus and Echinococcus multilocularis were new species in the studied area. The trematode Apophallus donicus , of which one individual was found by Borgsteede [ 4 ] was not identified in the present study.
This was also the case for Hymenolepis spp. Adult Hymenolepids are regarded as passing species from prey, as is Molineus patens , and these were thus excluded from analysis of helminth species parasitic to red fox. All foxes were negative for this species by microscopical examination of mucosal scrapings, but one fox out of investigated foxes was positive for E.
This positive result was confirmed after repeated testing of the faecal content. Up to this study, no positive foxes were identified in the presently studied area. Microscopic identification of cestodes was confirmed by cluster analysis of the partial CO1 gene sequences. The inferred Neighbour Joining tree shows very high homology between obtained CO1 sequences and Genbank entries for T.
Alaria alata is used as outgroup and here too, the Dutch isolates show high homology with other European isolates from Genbank. Bar indicates base substitutions per site. We report four new records of veterinary importance: Toxascaris leonina , Mesocestoides litteratus , Trichuris vulpis and Angiostrongylus vasorum.
Analysis of helminth species number Seventeen helminth species were identified from our reference data set of foxes. Epidemiological studies on intestinal helminth parasites of rural and urban red foxes Vulpes vulpes in the United Kingdom. Chapter 13 Volume P. Trends en broedresultaten van roofvogels in Nederland in Raptor breeding succes in The Netherlands in [in Dutch with summary in English] Takkeling. Dan Rev Game Biol. Above capacity of 3, ton BX-??
The finding of a fifth zoonotic species — Echinococcus multilocularis — has been described earlier for the Netherlands [ 20 ], but not in this same geographical area. We used a combination of microscopic and molecular techniques to evaluate the helminth fauna of red fox as described above, whereas Borgsteede [ 4 ] and Lucius et al.
Use of the more sensitive PCR technique in this present study might have biased the observed biodiversity to some extent, since it was not available in the period of the study of Borgsteede [ 4 ], but this does not explain the observed biodiversity increase compared to older studies. Confirmation of the identity of cestode species that had been found microscopically by PCR in this present study, did not lead to more cestode species compared to historic data. Moreover, even without E.
The introduction of E. The study of van der Giessen et al. This finding confirmed the observation of Borgsteede [ 4 ] at that time. Parasites indicated as Capillaria spp. Cremers, unpublished data , and other species passing through the gut after predation; however these were not further identified to species level.
Rarefaction and extrapolation of parasite richness and abundance data this study revealed a significant increase of species richness compared to 12 different fox parasite species determined by Borgsteede [ 4 ], 11 species found by Lucius et al. Recent studies in the Northern European hemisphere [ 6 , 8 ] show species richness that fits the asymptotic maximum of the estimated species richness calculated from our data. This increase might be driven by a combination of natural developments and or anthropogenic causes global warming, climatic fluctuations.
It is however, beyond the scope of this paper to identify the drivers for the observed increase in the parasite biodiversity.
Parasites of veterinary importance may be introduced into the environment through pet travel or translocation of wildlife hosts. Angiostrongylus vasorum only recently became endemic to the Netherlands [ 41 ] and is known for its endemic foci in Dutch dogs [ 41 ]. In the present study, we found A. In this study, E. The identification based solely on molecular techniques suggests a very low intestinal abundance in the infected fox, well below the detection level of microscopy. Previous studies showed PCR to be more sensitive, compared to the mucosal scraping method, especially at low endemicity [ 20 , 42 ].
However, this is not recognised in prevalence of patent infections in dogs [ 44 - 47 ]. The prevalence of Taenia spp. Among fox prey are rodents, which are obligate intermediate hosts in the lifecycle of cestode parasites like E. The decreasing prevalence of Taenia spp. We were able to identify Taenia crassiceps and T. CO1 gene sequences of A.
Previously, spatial prevalence analysis across borders demonstrated radiation of E. In the same period, the prevalence of two zoonotic helminths species belonging to different genera declined. In addition, four veterinary-important species were identified for the first time in this present study, and three additional species showed higher prevalence over that period. We identified the fox tapeworm E. Due to the very low prevalence and abundance, the infection risk for humans in the studied area is considered limited.
It remains important, however, to follow the spread of E. FF generated and analysed parasitological data, performed molecular lab work and sequence analysis, and wrote the manuscript, RN generated parasitological data and wrote the manuscript, JM generated biological data concerning the collected foxes, HC generated parasitological data concerning non-intestinal helminths, CD did the molecular lab work concerning E.
All authors read and approved the final manuscript. The authors thank Merel Langelaar, Marieke Opsteegh and Manoj Fonville for their valuable contribution to the study coordination, dissection of foxes and the parasitological examinations. National Center for Biotechnology Information , U. Journal List Parasit Vectors v. Published online Apr 3. Herman Cremers 4 Dr. Received Dec 12; Accepted Mar This article has been cited by other articles in PMC.
Abstract Background The red fox Vulpes vulpes is host to a community of zoonotic and other helminth species. Conclusions Helminth fauna in Dutch red foxes increased in biodiversity over the last three decades. Background Long-term studies on parasite communities of marine and terrestrial wildlife hosts were instrumental to evaluating the influence of natural and anthropogenic factors on environmental changes, especially when sampling series span more than ten years [ 1 - 3 ].
Open in a separate window. Microscopical examination of parasites Small intestine mucosal scraping The small intestine of each fox was separated and opened. Screening for cardio - pulmonary helminths The lungs and hearts of 97 foxes were examined for helminths by opening the right heart and pulmonary arteries up to the level of small branches in the lungs [ 34 ]. Screening for helminths in the urinary bladder In addition, four urinary bladders were opened to look for adult worms of Pearsonema plica.
Helminth species number To evaluate a possible change in helminth species richness, we applied rarefaction analysis [ 25 , 26 ] to the number of distinct helminth species that we identified in foxes. Results Animal age , gender and body weight In total, foxes were collected. Analysis of helminth species number Seventeen helminth species were identified from our reference data set of foxes. Multiple infections per fox On average Table 1 Overview of parasitic helminths found in Dutch red fox.