Molecular detection of Anaplasma phagocytophilum in wild carnivores in north-eastern Poland

Background Anaplasma phagocytophilum is an obligate parasitic intracellular bacterium. It is the causative agent of granulocytic anaplasmosis, with effects on human and animal health. In Europe, the pathogen is mainly transmitted among a wide range of vertebrate hosts by blood-sucking arthropods. The aim of this study was to determine the presence of A. phagocytophilum in wild carnivores, viz raccoon dogs (Nyctereutes procyonoides), badgers (Meles meles), foxes (Vulpes vulpes), martens (Martes sp.) and European polecats (Mustela putorius), using molecular methods. Methods In the present study, 174 spleen samples were collected from adult, wild carnivores hunted in the years 2013–2016. A short fragment (383 bp) of the 16S ribosomal RNA gene partial sequence was used as a marker to identify A. phagocytophilum in spleen samples collected from carnivores using nested PCR. Results The prevalence of A. phagocytophilum in wild carnivores was 31.61% (55/174). Seven sequences of A. phagocytophilum were generated from two raccoon dogs, two badgers, one marten, one red fox and one European polecat. Six identical nucleotide sequences were obtained from one raccoon dog, two badgers, one marten, one red fox and one European polecat (A. phagocytophilum sequences 1: MH328205–MH328209, MH328211), and these were identical to many A. phagocytophilum sequences in the GenBank database (100% similarity). The second sequence (A. phagocytophilum sequence 2: MH328210) obtained from the raccoon dog shared 99.74% identity with A. phagocytophilum sequence 1. Conclusions To our knowledge, this is the first study to use molecular methods to determine the presence of A. phagocytophilum in wild carnivores, viz raccoon dog, badger, marten and European polecat, in Poland. The detected A. phagocytophilum sequences (1 and 2) were closely related with those of A. phagocytophilum occurring in a wide range of wild and domestic animals and vectors.


Background
Anaplasma phagocytophilum is a Gram-negative bacterium belonging to the family Anaplasmataceae [1]. It is the causative agent of tick-borne fever in ruminants, also known as bovine or ovine granulocytic anaplasmosis, and of human granulocytic anaplasmosis. Anaplasma phagocytophilum is typically detected in wild animals (such as rodents, carnivores and ungulates) and domestic animals (such as cattle, goats and sheep) in the natural environment [2][3][4][5][6]. In the northern hemisphere, A. phagocytophilum is transmitted mainly by hard ticks of the genus Ixodes [7,8]. Species of other tick genera, such as Dermacentor spp. and Rhipicephalus spp., can also serve as biological vectors; however, their significance currently remains unknown. In addition, while A. phagocytophilum is known to demonstrate transstadial transmission in
Anaplasma phagocytophilum has also been detected in other arthropods such as blood-sucking flies, specifically deer flies (Lipoptena cervi) and horse flies (Diptera: Tabanidae) [9]. In addition, wild carnivores can also play a key role in pathogen transmission between wildlife, domestic animals and, possibly, humans. The growing degree of contact between free-living animals, domestic animals and human populations due to climate change and population growth has resulted in an elevated risk of human and bovine anaplasmosis outbreaks [10].
Knowledge about A. phagocytophilum infection in badgers, polecats and martens is still limited. It has been detected in European polecats (Mustela putorius) in Germany [22] and in the Netherlands [23], as well as in badgers (Meles meles) in the Netherlands and Spain [23,24]. It has also been identified among pine martens (Martes martes) in Belgium. [23].
With the exception of foxes, data regarding the levels of A. phagocytophilum infection in many wild carnivores, such as raccoon dogs, European polecats, martens and badgers, is scarce in Poland [25]. While the majority of these carnivores are native to central Europe, the raccoon dog (Nyctereutes procyonoides) is an introduced species from Asia. In Poland, they were first identified in the Białowieża Primeval Forest in 1955 [26]; they are currently widespread throughout central Europe, and their range is extending into western Europe [27].
These wild carnivores could potentially act as vectors of A. phagocytophilum in the natural environment [8]. The aim of the present study was therefore to determine the prevalence of A. phagocytophilum in five species of predatory wild animals in Poland. Molecular detection of A. phagocytophilum was based on semi-nested PCR amplification of the partial 16S rRNA gene according to Werszko et al. [9]. Three primers were used to molecular detection of A. phagocytophilum: A480F, A520F and A900R. The first PCR reaction was performed with primers A480F and A900R. For the second reaction (semi nested PCR), 1 μl of the first reaction product and primers A520F and A900R were used. PCR reactions were conducted in 50 µl of reaction mixture containing the following: 40 μl of deionized water in first reaction (41 μl in second), 1 μl of a 25 mM solution of MgCl 2 , 0.5 μl of Allegro Taq DNA Polymerase (5 U/μl; Novazym, Poznań, Poland), 0.5 μl of dNTPmix (10 mM), 5 μl of 10× Taq DNA Polymerase Buffer (with 25 mM MgCl 2 ), 0.5 μl of each primer (20 pmol/μl), 2 μl of template DNA in the first reaction and 1 μl of first reaction PCR product in the second reaction. DNA isolated from red deer (Cervus elaphus) infected with A. phagocytophilum (GenBank: GQ450278) was used as the positive control. Nuclease-free water was added to the PCR mix as a negative control. The PCR reaction was performed in an automated DNA Engine PTC-200 Peltier Thermal Cycler (BioRad, Hercules, CA, USA). The thermocycling profile was as follows: denaturation at 92 °C for 2 min, followed by 35 cycles with 30 s denaturation at 94 °C, 10 s primer annealing at 60 °C, and 1 min at 72 °C for primer extension, with a final extension step of 5 min at 72 °C.

Methods
The PCR products were subjected to electrophoresis (Bio-Rad Power Pac Basic 85 V, 45 min) on a 1.0% agarose gel stained with ethidium bromide and visualized using ChemiDoc, MP Lab software (Imagine, BioRad); a Nova 100 bp DNA Ladder (Novazym, Poznań, Poland) was used for comparison. The PCR amplicons were purified using a QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany), sequenced by Genomed (Warszawa, Poland) and assembled into contigs using ContigExpress, Vector NTI Advance v.11.0 (Invitrogen Life Technologies, New York, NY, USA). The obtained sequences were compared with sequences available on GenBank using the Basic Local Alignment Search Tool (BLAST). Statistical analyses were performed using the Chi-square test in Graph-Pad online (https ://www.graph pad.com).
Seven sequences were obtained from A. phagocytophilum PCR-positive samples and submitted to the Gen-Bank database (from two raccoon dogs, two badgers, one marten, one red fox and one European polecat). Six sequences (Anaplasma phagocytophilum sequence 1: MH328205-MH328209, MH328211) were found to be identical (100% similarity) with many A. phagocytophilum sequences obtained from Europe, Asia, Africa and North America. Anaplasma phagocytophilum sequences were obtained from a wide range of hosts: human, dog, raccoon dog, fox, sheep, black-striped field mouse, northern red-backed vole, tick, cow, goat and horse. More detailed data are shown in Table 1. Anaplasma phagocytophilum sequence 2 was highly similar to all examples of A. phagocytophilum sequence 1 obtained in this study (99.74% identity) and differed only by one nucleotide from sequence 1.

Discussion
Studies performed in Europe confirm that a wide range of mammals can serve as competent animal reservoirs for A. phagocytophilum, and that the composition of this reservoir varies according to geographical regions [8,28]. The high prevalence of A. phagocytophilum infection in the tested animals provides compelling evidence for the involvement of wild carnivores in the enzootic cycle. So far in Europe, A. phagocytophilum has been detected in red foxes in Germany (8.20%), Italy (16.60%) and Hungary (12.50%) [11,13,16]. It has also been detected in Poland, but only with an infection rate of 2.70% [25]. The higher prevalence identified in the present study (34.48%) may be associated with infection rate in the vectors [29], or the geographical distribution: the previous study was performed in central Poland, whereas the present study was performed in the north-east. Additionally, the prevalence was found to be 2.7% in the earlier study in Poland [25] which is close to prevalence levels obtained after the first PCR reaction in presented study. Anaplasma phagocytophilum can be detected in many various organs such as spleen, lung and liver tissue [4,12,13]. Our present findings confirm that semi-nested PCR offers a high level of accuracy in detecting Anaplasma in spleen samples, and that it is more sensitive than standard PCR: while the detected prevalence was only 4/174 (2.30%) in all tested animals after the first reaction, the detection level was increased to 55/174 (31.60%) after the second stage.
Foxes are well adapted to the urban environment and are accustomed to the presence of humans. In addition, their frequent exposure to tick bites and their potential to act as reservoirs or maintenance hosts for pathogens infecting humans and domestic animals highlights their importance in public health [15]. The prevalence of A. phagocytophilum observed in raccoon dogs in Poland (35.30%) is higher than in Germany (23.00%) [13]. Interestingly, while Han et al. [21] and Kang et al. [30] reported the prevalence of A. phagocytophilum infection in this host to be 1.00% in Korea, Hildebrandt et al. [31] reported no infection in raccoon dogs in western Poland. According to the present findings, 18.70% of the tested badgers in north-eastern Poland displayed A. phagocytophilum infection; however, Garcia-Pérez et al. [24] reported a prevalence of 1.50% among badgers in Spain, and Hofmeester et al. [23] found it to be 1.80% in the Netherlands. While Anaplasma infection was found in two of the four tested European polecats in the present study, previous studies have found the prevalence to be 4.30% in Germany and 4.90% in the Netherlands [22,23]. Similarly, A. phagocytophilum was found to be more common in pine martens (Martes martes) in the present study (41.70%) than in Belgium (22.00%) [23].

Conclusions
All five tested species of carnivore may serve as suitable reservoir hosts in the ecology of Anaplasma phagocytophilum in the natural environment. Most isolates obtained in this study were identical to A. phagocytophilum sequences deposited in GenBank. Our findings represent the first detection of A. phagocytophilum in badgers, raccoon dogs, martens and polecats from Poland. The nested PCR used in the present study was more sensitive to detection of A. phagocytophilum than standard PCR for spleen tissue samples.