Role of invasive carnivores (Procyon lotor and Nyctereutes procyonoides) in epidemiology of vector-borne pathogens: molecular survey from the Czech Republic

Abstract

Background: Vector-borne pathogens (VBPs) are a major threat to humans, livestock and companion animals worldwide. The combined effect of climatic, socioeconomic and host composition changes favours the spread of the vectors, together with the expansion of invasive carnivores contributing to the spread of the pathogens. In Europe, the most widespread invasive species of carnivores are raccoons (Procyon lotor) and raccoon dogs (Nyctereutes procyonoides). This study focused on the detection of four major groups of VBPs namely Babesia, Hepatozoon, Anaplasma phagocytophilum and Bartonella in invasive and native carnivores in the Czech Republic, with the emphasis on the role of invasive carnivores in the eco-epidemiology of said VBPs.

Methods: Spleen samples of 84 carnivores of eight species (Canis aureus, Canis lupus, Lynx lynx, P. lotor, Martes foina, Lutra lutra, Mustela erminea and N. procyonoides) were screened by combined nested PCR and sequencing for the above-mentioned VBPs targeting 18S rRNA and cytB in hemoprotozoa, groEL in A. phagocytophilum, and using multilocus genotyping in Bartonella spp. The species determination is supported by phylogenetic analysis inferred by the maximum likelihood method.

Results: Out of 84 samples, 44% tested positive for at least one pathogen. Five different species of VBPs were detected in P. lotor, namely Bartonella canis, Hepatozoon canis, Hepatozoon martis, A. phagocytophilum and Bartonella sp. related to Bartonella washoensis. All C. lupus tested positive for H. canis and one for B. canis. Three VBPs (Hepatozoon silvestris, A. phagocytophilum and Bartonella taylorii) were detected in L. lynx for the first time. Babesia vulpes and yet undescribed species of Babesia, not previously detected in Europe, were found in N. procyonoides.

Conclusions: Wild carnivores in the Czech Republic are hosts of several VBPs with potential veterinary and public health risks. Among the studied carnivore species, the invasive raccoon is the most competent host. Raccoons are the only species in our study where all the major groups of studied pathogens were detected. None of the detected pathogen species were previously detected in these carnivores in North America, suggesting that raccoons adapted to local VBPs rather than introduced new ones. Babesia vulpes and one new, probably imported species of Babesia, were found in raccoon dogs.

Keywords: Anaplasma phagocytophilum; Babesia; Bartonella; Carnivores; Hepatozoon; Invasive species; Vector-borne pathogens.

Fig. 1

Schematic representation of the maximum likelihood phylogenetic tree based on the 18S rRNA gene sequences of Babesia s.s. of a dog-infecting subclade. The final length of the alignment was 1750 bp and the tree was constructed using the evolution model TPM3+F+R2. Three sequences of B. caballi, the closest species, used as an outgroup are not displayed. The clade in a sister position to B. honkongensis is shown in detail. Sequences from this study are marked in red. The scale bars indicate the number of nucleotide substitutions per site. The bootstrap values (SH-aLRT/UFB) above the 80/95 threshold are displayed. Sequences are labelled by accession number, host and country of origin (if available)

Fig. 2

Schematic representation of the maximum likelihood phylogenetic tree based on the 18S rRNA gene sequences of the suborder Adeleorina, focused on a well-defined clade of mammal-infecting Hepatozoon spp. The final length of the alignment was 2139 bp and the tree was constructed using the evolution model GTR+F+R4. Two sequences of Hammondia hammondi and one sequence of Toxoplasma gondii used as an outgroup are not displayed. The cluster of clades containing sequences of H. martis, H. silvestris, H. felis and H. ursi is shown in detail. Sequences from this study are marked in red. The scale bars indicate the number of nucleotide substitutions per site. The bootstrap values (SH-aLRT/UFB) above the 80/95 threshold are displayed. Sequences are labelled by accession number, host and country of origin (if available)

Fig. 3

Schematic representation of the maximum likelihood phylogenetic tree based on the groEL gene sequences of Anaplasma phagocytophilum. The final length of the alignment was 1256 bp and the tree was constructed using the evolution model TN+F+I+R2. Three sequences of A. platys used as an outgroup are not displayed. Detailed clades belonging to ecotype I (A) and ecotype II (B) as described by Jahafari et al. (2014) are shown. Sequences from this study are marked in red. The scale bars indicate the number of nucleotide substitutions per site. The bootstrap values (SH-aLRT/UFB) above the 80/95 threshold are displayed. Sequences are labelled by accession number, host and country of origin (if available)