Under the Skin: Ixodes Ticks in the Subcutaneous Tissue of Red Foxes
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Haut et al. Parasites Vectors (2020) 13:189 https://doi.org/10.1186/s13071-020-04061-x Parasites & Vectors SHORT REPORT Open Access Under the skin: Ixodes ticks in the subcutaneous tissue of red foxes (Vulpes vulpes) from Germany Maja Haut1, Nina Król1, Anna Obiegala1, Johannes Seeger2 and Martin Pfefer1* Abstract Background: Ixodes spp. are vectors of zoonotic pathogens. All three active life stages (larvae, nymphs, adults) need to feed on a host in order to develop. Usually ticks parasitize attached to the external surface of their hosts’ skin. Interestingly, in some cases ticks can also be found in the subcutaneous tissue in a variety of hosts, such as red foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and dogs. Methods: The visceral side of 126 red fox-furs from Germany was examined visually searching for ticks. The localiza- tion of ticks was recorded and assigned to ten specifc body parts. Morphological identifcation of ticks was per- formed according to standardized taxonomic protocols. Ticks which could not be further identifed were examined genetically via conventional PCR targeting the 16S rRNA and cox1 gene. Hematoxylin and eosin (H&E) staining was used for histopathological examination. Results: In 111 out of 126 (88.1%) examined coats, at least one tick was found in the subcutaneous tissue. A total of 1203 ticks were removed from the subcutaneous tissue. Well-preserved ticks could be identifed based on morpho- logical criteria, but most ticks were in a progressed state of decomposition. Here, morphological species identifcation was not successful. Also, PCR methods did not lead to a successful species identifcation. The following species and development stages were found by morphological identifcation: Ixodes ricinus (female, n 289; male, n 8; nymph, n 1), I. hexagonus (female, n 2), I. canisuga (female, n 1). Male I. ricinus were found individually= or copulating= in pairs= with females. Subcutaneous= ticks were localized at= three predominant afected body parts: ears, axillar and ingui- nal region. Histological examination of subcutaneous ticks revealed a granulomatous panniculitis. Conclusions: To the authors’ knowledge, this is the frst fnding of highly prevalent subcutaneous ticks in red foxes from Germany. Subcutaneous location of ticks seems to be very common in red foxes and the rule rather than the exception. Deep embedment of longirostra and long feeding times of females seem to put the subcutaneous loca- tion in favor. Most foxes were infested in the inguinal area, where the skin is thin and less hairy. Keywords: Ectoparasites, Subcutaneous, Ixodes spp., Tick, Red fox, Germany, Europe Background Ticks belonging to the genera Ixodes and Dermacentor (Family: Ixodidae) are the most prevalent in Europe [1]. All active tick life stages (six-legged larva, eight-legged *Correspondence: [email protected] nymph and females) require blood meals from hosts for 1 Institute of Animal Hygiene and Veterinary Public Health, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 1, molting (immatures) or for oviposition (females) with the 04103 Leipzig, Germany exception of males which feed facultatively and are local- Full list of author information is available at the end of the article ized on hosts mainly for mating purposes [1, 2]. © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Parasites Vectors (2020) 13:189 Page 2 of 9 In Germany the species Ixodes ricinus, I. hexagonus, from all over Germany deposited fox carcasses in plas- I. canisuga and I. kaiseri have been frequently collected tic bags labelled with the date and exact geographical from foxes [3–8]. Additionally, Dermacentor reticu- location in deep-freezers. Only adult foxes with good latus and Haemaphysalis concinna were occasionally nutritional status were selected by hunters. Fox cadav- found attached to red foxes’ skin [7, 9]. Ticks are usu- ers were stored at − 80 °C until faying. For enabling the ally strongly attached to the external surface of their histological studies one fox was skinned directly after hosts to remain in place for a blood meal. Tereby tick’ hunting. mouthparts penetrate diferent skin layers, depending on their length. Ticks with long mouthparts (longirostra Tick collection and identifcation e.g. Amblyomma, Hyalomma and Ixodes) embedded into Te visceral side of each fur was examined visually for deep layers of the dermis while ticks with short mouth- the presence of subcutaneous ticks. Te localization of parts (brevirostra e.g. Dermacentor, Haemaphysalis and ticks was recorded on a pre-drawn schematic picture and Rhipicephalus) barely penetrate the epidermis [10, 11]. assigned to ten specifc body parts (ears, neck, axillar Ixodes ricinus larvae are attached for 2–4 days, nymphs region, shoulder, back, belly, inguinal region, anorectal for 4–6 days and females for 6–10 days [12]. region, legs and tail) in order to create a body map of tick Interestingly, in some cases ticks can also be found in density (Fig. 1), as recommended by Lydecker et al. [25]. the subcutaneous tissue in a variety of hosts including Afterwards, the ticks were individually dissected from red foxes. Te presence of subcutaneous ticks such as I. the subcutaneous tissue with fne-tipped forceps and ricinus, I. hexagonus, I. crenulatus and D. reticulatus was a scalpel and stored at − 20 °C (Fig. 2). Well-preserved noted in red foxes in several case studies from the UK, ticks could be identifed based on morphological crite- Poland, Austria, Romania, Slovakia and Czech Republic ria according to standardized taxonomic protocols [26] [13–20]. In the USA, the native tick species Amblyomma under a binocular at 50× magnifcation (Motic SMZ-171, americanum was likewise discovered in the subcutaneous Motic Deutschland GmbH, Wetzlar, Germany). tissue of red foxes [21]. Besides red foxes, single case reports of subcutaneous DNA extraction and PCR methods ticks were previously recorded in raccoon dogs (Nyc- Ticks which could not be morphologically identifed were tereutes procyonoides) from Poland [22], in a dog from further processed by conventional PCR to identify them Sweden [23] and in a child from South Korea [24]. to species level. For the DNA extraction, 1 g of steel beads Hitherto, small sample sizes were reported in most (sized 2.8 mm) and 500 µl of PBS were added to each tick. cases also rather as occasional fndings [13–17, 21]. Te samples were then homogenized at 5000× rpm for Tus far, no published data exist about subcutaneous ® 2 × 15 s with 15 s break intervals in a Precellys 24 tissue ticks in red foxes or other animals from Germany. Tis homogenizer (Bertin Technologies, Montigny Le Breton- is why this study is aiming to evaluate the frequency of neux, France). subcutaneous ticks in red foxes from Germany using Te genomic DNA was extracted using the QIAamp a large number of samples. Te localization of subcu- DNA Mini Kit (Qiagen, Hilden, Germany) following the taneous ticks at diferent body regions of the red fox manufacturer’s instructions. PCR amplifcations of par- has not been analyzed yet; therefore, we documented tially cytochrome c oxidase subunit 1 (cox1) gene (710 the localization of each tick in order to determine the bp) and of the 16S ribosomal ribonucleic acid (rRNA) abundance of these ticks in the subcutaneous tissue. gene (455 bp) were performed using previously published Precise species identifcation of subcutaneous ticks PCR protocols [8, 27]. PCR products were visualized based on morphological criteria is possible only to a by gel electrophoresis on 2% agarose gels stained with limited extent due to the advanced degradation process HDGreen Plus DNA Stain (Intas Science Imaging Instru- in subcutaneous tissue, as seen in previous studies [18]. ments GmbH, Göttingen, Germany). Hence, this study aims to use molecular biology tech- niques to identify subcutaneous ticks to species level. Histological examination of subcutaneous tissue Subcutaneously located ticks were removed with the skin and the surrounding tissue from the complete fur and Methods immediately fxed in 10% bufered formalin, dehydrated Animal collection in ascending ethanol series (20–100%) and embedded Between November 2018 and February 2019 local in parafn blocks. Seven-micron thick sections were hunters shot red foxes and other wild game in course cut with a rotary microtome (Leica RM 2155, Leica of traditional hunting. In cooperation with a scrape Microsystems Nussloch GmbH, Nussloch, Germany), station (Fellwechsel Berlin, Germany), local hunters placed on glass slides and stained with hematoxylin and Haut et al. Parasites Vectors (2020) 13:189 Page 3 of 9 Fig. 1 Density of ticks expressed as percentage of total number of ticks depending on body region (n 111 red foxes). A darker grey in the fgure = indicates a higher density of ticks eosin (H&E). Microscopy and pictures were performed by the Clopper & Pearson method with GraphPad Soft- using a Zeiss Axioplan 2 imaging (Carl Zeiss Microscopy, ware (GraphPad Software Inc., San Diego, CA, USA).