The Occurrence of Zoonotic Anaplasma phagocytophilum Strains, in the Spleen and Liver of Wild Boars from North-West and Central Parts of Poland

Purpose The Anaplasma genus includes a Gram-negative bacterium infecting the blood cells of wild and domestic mammals, causing tick-borne fever. Infection with pathogenic Anaplasma phagocytophilum strains may cause Human Granulocytic Anaplasmosis. Wild boars (Sus scrofa) may act as natural wild reservoir hosts for potentially zoonotic A. phagocytophilum strains; however, there is still little data to confirm this statement. The aim of this study was to verify whether wild boars can be classified as natural reservoirs of Anaplasma spp. and to compare the suitability of spleen and liver samples for such analysis. Methods Liver and spleen samples were collected from 59 wild boars (2017–2019). The organs were tested for Anaplasma phagocytophilum using short (partial) fragments of three markers: 16S rRNA, groEL, ankA. Results Anaplasma spp. DNA was detected in 12 out of 59 samples, with a prevalence of 20.34%. The presence of A. phagocytophilum was confirmed by sequencing of the partial 16S rRNA gene. Positive individuals were tested for the characteristic markers: groEL and ankA. The analysis of the nucleotide sequences of 16S rRNA, groEL and ankA, indicated that the strains of A. phagocytophilum detected in these studies are potentially zoonotic for humans. Conclusion Wild boars from Poland can be classified as a natural reservoir of the zoonotic strain of Anaplasma phagocytophilum. Both the spleen and the liver tissues were found to be suitable materials for the detection of A. phagocytophilum.


Introduction
The causative agent of Human Granulocytic Anaplasmosis (HGA), tick-borne fever (TBF) and granulocytic anaplasmosis in wild, domestic and farm animals is the parasitic bacterium Anaplasma phagocytophilum [1,2]. Anaplasma spp. are gram-negative bacteria with a specific cell wall structure. Anaplasma phagocytophilum is an obligatory intracellular parasite that lives in neutrophils [3] and can be transmitted by ticks: Ixodes ricinus, I. persulcatus, I. scapularis and Dermacentor reticulatus [4][5][6]. Recent reports by Werszko et al. (2019) [7] show that the blood-sucking flies from Tabanidae family can act as carriers of A. phagocytophilum; however, more research is needed to confirm whether they can act as transmission vectors. Ticks and other hematophagous ectoparasites can easily transfer such bacterial intracellular parasites between natural animal reservoirs and humans [8,9]. The considerable climate changes observed over the past 20 years have resulted in the spread of arthropods, such as ticks and flies, carrying pathogenic bacteria into new regions. Such an increase in annual temperatures has also led to a significant increase in the number of ectoparasites already present in the area, which significantly influences the spread of intracellular bacterial parasites, including Anaplasma spp., in the natural environment [10]. Many wild animals, such as roe deer (Capreolus capreolus), red deer (Cervus elaphus), wild boar (Sus scrofa), red fox (Vulpes vulpes), raccoon dog (Nyctereutes procyonoides) and the European badger (Meles meles) are infected with A. phagocytophilum [11,12]. Wild boars are likely to be natural wild reservoir hosts for potentially zoonotic A. phagocytophilum strains; however, there is still little data to confirm this statement [11,13].

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The aim of this study was to verify whether the wild boar populations in the north-east and central parts of Poland can be natural reservoirs of A. phagocytophilum, a bacterium potentially pathogenic to humans. It also determines which of the internal organs collected from wild boar, i.e., spleen or liver, is more suitable for the detection of Anaplasma phagocytophilum.

Materials and Methods
All materials were collected during the 2017/2018 hunting season in the Pisz Forest (Warmian-Masurian Voivodeship) and in the 2018/2019 hunting season in the Bolimów Forest (Łódź Voivodeship). In total, spleen and liver samples were collected from 59 adult wild boars.
DNA from both organs was isolated using a commercial DNA Mini Kit (Syngen). Anaplasma spp. was then detected using semi-nested PCR to amplify the partial 16S rRNA gene with primers specific to Anaplasma genus according to Szewczyk et al. (2019) [12]. Positive samples for A. phagocytophilum were additionally tested for the presence of the partial groEL and ankA genes with nested PCR according to Alberti el al. (2005), Massung et al. (2007) and Rymaszewska (2014), respectively (Table 1) [14][15][16]. DNA amplification was performed using the DNA Engine T100 Thermal Cycler (BioRad, USA). The PCR products were visualized on a 1.2% agarose gel (Promega, USA) stained with Sim-plySafe (EURx, Poland). Visualization was performed using ChemiDoc, MP Lab software (Imagine, BioRad, USA). The obtained PCR products were purified with the QIAquick Purification Kit (Qiagen, Germany). The purified products were sequenced directly using ABI BigDye™ chemistry (Applied Biosystems, USA) on an ABI Prism 373xl or an ABI Prism 3100™ automated sequencer. The obtained sequences were submitted to the GenBank.

Results and discussion
Of the 59 wild boar from which spleen and liver samples were taken, DNA of Anaplasma spp. were detected in 12 individuals, i.e., a prevalence of 20.34%. Anaplasma spp. genetic material was detected in seven individuals in the spleen samples, and in six individuals in the liver samples ( Table 2). All positive samples were obtained from boars in the Pisz Forest; no positive samples were found in the Bolimów Forest. Four positive samples were selected for sequencing, and the results indicated the presence of A. phagocytophilum.
A number of studies in various countries have been performed on wild boars with the aim of identifying natural reservoirs of zoonotic strains of A. phagocytophilum [2,17,18]. Most of these tests are based on analyses of blood and spleen samples [1,2,17,19,20] and, very rarely, liver samples [18,21]. Although some individual studies have used both the spleen and liver [21,22] none indicate which is more suitable for this type of analysis. Our findings clearly show that, in wild boars, both these tissues are suitable (Table 2). However, as only one examined individual demonstrated positive results for both organs (1/59), it is advisable that both organs should sampled to maximize the detection possibilities when there is no access to blood, which is the best material for this type of analysis [23].
The prevalence of Anaplasma phagocytophilum in wild boars varies across Europe and elsewhere, ranging from 0.97% in Belgium [24] to 44.8% in France [13]. In addition, one study reports that genetic material of A. phagocytophilum was not detected in the tested wild boar in Slovakia [25].
Comparing the prevalence of A. phagocytophilum in wild boars in three regions of Poland, it can be seen that it is much more widespread in the west-central (12%) [18] and north-central (20.34%) regions than in the central (no positive samples) region (this study). The higher prevalence in the west-central and north-central regions may be due to the high afforestation density [28], which favors an increased incidence of ticks, these being known vectors of A. phagocytophilum [29]. The presence of increased numbers of vectors in an environment enables a faster spread of A. phagocytophilum among hosts. A similar correlation between geographic distribution and an increase in host prevalence has been shown by Szewczyk et al. [12].
Our sequencing of selected positive 16S rRNA partial gene samples (n = 4) confirmed the presence of A. phagocytophilum in the tested wild boars (MT510541 The two most common markers used to describe the genetic diversity of A. phagocytophilum strains are the groEL and ankA genes [10]. Based on our analysis of the groEL gene sequences (MT731760, MT731761, MT731762) the detected strains of A. phagocytophilum were classified into ecotype I [10,30]. In the groEL gene, one nucleotide sequence (MT731760) showed 100% similarity to a groEL sequence from the human strain of A. phagocytophilum (AF033101), while another two (MT731761, MT731762) showed only 99.78%. However, despite the point change observed in the latter two sequences, all three encode the same protein as in human strains of A. phagocytophilum. Regarding ankA, two sequences (MT534241, MT731758), obtained from two wild boar individuals, are 100% complementary to human isolates (AF100886, AF100887, GU236800). The third isolate from wild boar (MT731759) showed 99.4% similarity to human markers. From all obtained ankA gene sequences, only two of them (MT534241 and MT731758) encode the same protein as human A. phagocytophilum strains: the third one (MT731759) is significantly different from the protein detected in humans. The analysis of the nucleotide sequences of 16S rRNA, groEL and ankA indicates that the strains of A. phagocytophilum detected in samples in this study can be classified as potentially zoonotic for humans.

Conclusion
Our findings suggest that wild boars from Poland can be classified as a natural reservoir of the zoonotic Anaplasma phagocytophilum strains. In addition, both the spleen and liver were found to be suitable for the detection of A. phagocytophilum. However, further research is needed in other areas of Poland to comprehensively analyze the issues of A. phagocytophilum natural reservoirs throughout the country, and such studies should include other animals, which may demonstrate different tissue predilection than wild boars.
Acknowledgements The authors would like to thank the management and staff of Strzałowo Forest District and Hunting Club No. 9 "Knieja" from Warsaw.

Author contributions
All authors contributed to the study conception and design. Material preparation was performed by: AWM, TS, data collection was performed by: AWM, TS, ZL and analysis were performed by: AWM, ZL. The first draft of the manuscript was written by AWM and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding
The authors received no financial support for the research, authorship, and publication of this article.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent All authors consent to the publication.
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