Anaplasma phagocytophilum in Multiple Tissue Samples of Wild Carnivores in Romania

Wildlife may play a major role in the transmission and maintenance of zoonotic agents, with most emerging infectious diseases being of wildlife origin (Jones et al. 2008). However, knowledge of the pathogens that naturally occur in wild animals, and their potential to spread to humans and domestic animals, is still scarce, especially for tick-borne pathogens (Tomassone et al. 2018). In Europe, tick-borne rickettsial infections include zoonotic diseases such as spotted fever group (SFG) rickettsioses, anaplasmoses, and ehrlichiosis (Parola 2004).

There are studies that suggest the involvement of vertebrate hosts in the epidemiology of SFG Rickettsia spp. (Tomassone et al. 2018). Among the wild carnivores, Rickettsia spp. DNA has previously been detected in red foxes (Vulpes vulpes) in Switzerland and Lithuania (Hofmann-Lehmann et al. 2016; Sakalauskas et al. 2019). Despite rickettsial DNA detection in wild carnivores, in a study from Spain, Rickettsia massiliae–positive ticks were collected from Rickettsia-negative carnivores (Millán et al. 2016), raising uncertainties regarding their reservoir role. Nevertheless, the biologic and social features of wild carnivores (large size, large home range, and long life span) suggest a possible importance as hosts for ticks and tick-borne pathogens, as these aspects allow cofeeding by different tick species, which may be an important transmission mechanism in perpetuating rickettsiae in nature. Moreover, high tick aggregation levels on a given individual may favor Rickettsia maintenance in tick populations (Tomassone et al. 2018).

Ehrlichia canis, a species with zoonotic potential, has been detected in red foxes, gray wolves (Canis lupus), raccoons (Procyon lotor), and Eurasian otters (Lutra lutra) in Italy, Portugal, and Spain (Cardoso et al. 2015; Millán et al. 2016; Santoro et al. 2017; Criado-Fornelio et al. 2018).

Compared to other rickettsial agents, Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, seems to be more common in wild carnivores. It has been detected in red foxes, brown bears (Ursus arctos), gray wolf, raccoon dogs (Nyctereutes procyonoides), European polecat (Mustela putorius), golden jackals (Canis aureus), European badger (Meles meles), raccoons, and stone martens (Martes foina) in several European countries (Víchová et al. 2010; Ebani et al. 2011; Härtwig et al. 2014; Tolnai et al. 2015; Hofmann-Lehmann et al. 2016; Jaarsma et al. 2019; Battisti et al. 2020). Although the role of wild carnivores as reservoir hosts in Europe is uncertain, some species such as raccoon dogs and red foxes are considered to be capable of maintaining A. phagocytophilum in nature (Härtwig et al. 2014). Anaplasma platys, a related pathogen with a suggested zoonotic potential (Maggi et al. 2013), has been detected in red foxes from Portugal (Cardoso et al. 2015).

We evaluated the presence of rickettsial agents in wild carnivores from Romania, considering their potential role as sentinel species for tick-borne diseases. In addition, motivated by the variable results in Anaplasma spp. detection and the infrequent detection of Rickettsia spp. in tissue samples, we also aimed to compare the presence of rickettsial DNA in different sample types.

The wild carnivores included in our study were collected from authorized hunters during the legal hunting period, and from rangers who found dead animals (e.g., road kills) during 2014–18, from 17 counties of Romania (Fig. 1). At necropsy, blood clots, heart, liver, lung, spleen, kidney, lymph node, and bone marrow were sampled for this study. In total, 760 samples were collected from 95 wild carnivores belonging to nine species (golden jackal, grey wolf, European wildcat, European otter, Eurasian lynx [Lynx lynx], stone marten, European badger, brown bear, and red fox; Fig. 1).

Genomic DNA was extracted for each tissue sample using the ISOLATE II Genomic DNA Kit (Bioline, Meridian Bioscience, Cincinnati, Ohio, USA), following the manufacturer's instructions. The quality and quantity of genomic DNA were evaluated using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA).

We used nested PCR to detect A. phagocytophilum and E. canis DNA in samples, using specific primers amplifying fragments of the rrs gene, while SFG Rickettsia DNA detection was performed using a group-specific set of primers amplifying a fragment of the rickettsial gltA gene (PCR reactions are in the Supplementary Material). In each PCR reaction set, positive and negative controls were included in order to assess the specificity of the reaction and the possible cross-contamination. Positive controls consisted of DNA extracted from a dog naturally infected with A. phagocytophilum and from a castor bean tick (Ixodes ricinus) infected with Rickettsia helvetica, both confirmed by sequencing. The PCR was carried out using a T100™ Thermal Cycler (Bio-Rad, Hercules, California, USA). Amplicons were visualized by electrophoresis in a 1.5% agarose gel stained with SYBR® Safe DNA gel stain (Invitrogen, Carlsbad, California, USA).

All positive PCR samples were sequenced at Macrogen Europe (Amsterdam, the Netherlands), and the obtained sequences were analyzed and compared with those available in GenBank™ by BLAST (National Center for Biotechnology Information, Bethesda, Maryland, USA) analysis.

Statistical analysis was performed using Epi Info™7 software (Centers for Disease Control, Atlanta, Georgia, USA). Pathogen prevalence and 95% confidence interval (CI), and the infection prevalence differentiated by tissue sample, were assessed using the chi-squared test for independence.

Amplification of E. canis and Rickettsia spp. DNA was negative in all tested samples. Anaplasma phagocytophilum was detected with an overall prevalence of 10.53% (10/95, 95% CI 5.16–18.51). In total, 13 positive tissue samples were detected in five golden jackals and in one each of grey wolf, European wildcat, stone marten, Eurasian otter, and brown bear (Table 1). Multiple positive samples were detected in golden jackal, Eurasian otter, and brown bear (Table 2). All samples from Eurasian lynx, red fox, and European badger were negative. The positive animals were collected from seven counties: golden jackals in Tulcea, Buzau, and Vâlcea counties, grey wolf in Mureş county, European wildcat in Maramureş county, stone marten in Tulcea county, Eurasian otter in Alba county, and brown bear in Braşov county (Fig. 1).

The highest prevalence of A. phagocytophilum DNA was detected in spleen tissue (6.32%, 95% CI 2.23–13.24), followed by bone marrow and kidney (2.11%, 95% CI 0.26–7.4), blood clot, liver, and lung (1.05%, 95% CI 0.03–5.73); A. phagocytophilum DNA of this pathogen was detected in the spleen of more than half of the positive animals. Only small numbers of samples were positive, and statistical analysis did not show significant differences between the prevalence differentiated by species, geographic origin, or tissue sample type.

Sequence analysis showed a high similarity (100%) among the sequences obtained from wild carnivores (Supplementary Material Fig. S1) and 99–100% similarity with European A. phagocytophilum strains (GenBank nos. CP006618 and JX173651).

Anaplasma phagocytophilum has not previously been reported in European wildcat and European otter. Few previous studies on A. phagocytophilum infection in wild carnivores in Europe have clearly specified the tissue samples that were used. In red foxes from Italy, Poland, and Hungary, the specific DNA was detected in spleen tissue (Karbowiak et al. 2009; Ebani et al. 2011; Tolnai et al. 2015). In Czech Republic, one red fox was found to be positive without any indication of the type of tissue sample that was positive (Hulínská et al. 2004). Similarly, in brown bears, the collected tissue was “muscle, liver or spleen,” without any data regarding the tissues in which A. phagocytophilum DNA was found (Víchová et al. 2010). Anaplasma phagocytophilum was also detected in the lungs of red fox and raccoon dog in Germany (Härtwig et al. 2014).

Based on our results and on the high prevalence in red foxes obtained in Italy and Hungary (Ebani et al. 2011; Tolnai et al. 2015), the spleen may be the most appropriate sample type for A. phagocytophilum screening. However, since this pathogen may also be detected in other tissues while spleen is negative, a mixture from multiple tissue samples may work better. Based on our results, positive results are less commonly found in heart and lymph node samples.

Several wild carnivores have been suggested as possible reservoir hosts for A. phagocytophilum in the US, while in Europe, based on the frequency of its detection, only red foxes are considered to be suitable reservoir hosts (André 2018). The phylogenetic analysis of the rrs DNA fragment sequences obtained in our study showed the relatedness with zoonotic strains. However, rrs DNA fragment sequence analysis does not have sufficient discriminatory power to classify these strains into biotypes or ecotypes. Based on previous studies, all wild carnivores seem to be infected with potential zoonotic strains belonging to ecotype I (Jaarsma et al. 2019). Thus, their importance in the A. phagocytophilum ecoepidemiology should be further investigated. The results of our study together with other relevant published papers indicate that wild carnivores may be involved in the ecoepidemiology of this pathogen by maintaining the infection in synanthropic environments.

We would like to acknowledge the help provided by Jana Ababii, Aikaterini Alexandra Daskalaki, and Attila D. Sándor in sample collection. This work was supported and funded by the CNCS-UEFISCDI Grant Agency Romania, grants PD 34/2018 and PCCDI 57/2018.

Supplementary material for this article is online at http://dx.doi.org/10.7589/JWD-D-20-00158.