Molecular characterization of Anaplasma phagocytophilum infection in the cervids and feeding ticks from Lithuania

INTRODUCTION

Anaplasma phagocytophilum is small gram-negative obligate intracellular bacterium, which is the main agent causing zoonotic diseases such as granulocytic anaplasmosis in humans (HGA) and animals (Nicholson et al., 2010; Hajdusek et al., 2013). A. phagocytophilum has a  broad geographic distribution and a high degree of biological and clinical diversity. A.  phagocytophilum was first recognized in Europe 80 years ago as a causative agent of tick-borne fever (TBF) in domestic ruminants. Ticks act as the  main vector of A.  phagocytophilum. A. phagocytophilum is mainly transmitted by ticks belonging to the Ixodes ricinus complex: Ixodes ricinus in Europe, Ixodes persulcatus in Eastern Europe and East Asia, and Ixodes scapularis and Ixodes pacificus in North America (Jaarsma  et  al., 2019). Infected ticks can transmit A. phagocytophilum to new hosts during the  blood meal of its following stage. Ixodes ticks can be infected by A.  phagocytophilum at each stage (except as eggs), but only nymphs and adult females can transmit this bacterium. Anaplasma bacteria infects a wide range of wild mammalian, domestic animals, rodents and humans. Two distinct A. phagocytophilum clades were detected based on four genes of bacteria genome: one clade contained A.  phagocytophilum genotypes from questing I. ricinus and feeding I. ricinus from a broad array of hosts (humans, ungulates, birds and dogs), while the other clade comprised solely genotypes found in rodents and feeding I. trianguliceps (Blaňarová et al., 2014).

The role of wildlife species in the circulation of A. phagocytophilum is yet to be clearly determined, but several species of wild ruminants are thought to be important reservoirs. Several species of wild ruminant have been suggested to act as reservoir hosts, amongst them mainly the roe deer and the red deer (Woldehiwet 2010; Stuen  et  al., 2013; Dugat  et  al., 2015). In Lithuania, the cervids such as the roe deer, the red deer, and the moose are important game animals but their contribution as reservoirs for A. phagocytophilum remains unknown. Epidemiological studies in multiple countries have shown that the prevalence of A. phagocytophilum highly depends on the density of ticks and their potential hosts such as the cervids, which are one of the main sources of nutrition for Ixodes ticks. Previous studies have also suggested that A. phagocytophilum strains circulating in different ruminant species, possibly having distinct transmission cycles in nature, are independent of each other (Massung et al., 2002; Stuen et al., 2003, 2013).

The objectives of the study were to investigate the prevalence of A. phagocytophilum infections in the  cervids and feeding ticks and to characterize the A. phagocytophilum strains obtained from the cervids and ticks based on sequence analysis of msp4 gene.

MATERIALS AND METHODS

Ticks were sampled from 44 cervids representing three species: the roe deer Capreolus capreolus, the red deer Cervus elaphus, and the moose Alces alces (Table  1) harvested by professional hunters during the  hunting seasons of 2010–2013 and 2016–2017 in nine districts of Lithuania (Biržai, Jonava, Jurbarkas, Kaunas, Rumšiškės, Tauragė, Ukmergė, Vievis, and Vilnius). Ticks were collected from hosts using tweezers and placed into 1.5 ml tubes with 70% ethanol. Blood or spleen samples were collected from 29 animals (27 roe deer and two red deer) hunted from 2010 to 2013.

Identification of tick species and determination of the  life stage was done microscopically based on morphological characters (Estrada-Peña  et  al., 2004). From partially or fully engorged ticks DNA was extracted using Genomic DNA Purification Kit (Thermo Fisher Scientific, Lithuania), while from unfed ticks DNA was extracted using a  modified procedure with the  ammonium hydroxide solution (2.5%) (Stańczak et al., 1999). The lysates were stored at –20°C until PCR analysis. The samples were screened for the presence of A. phagocytophilum by nested PCR as described by de la Fuente et al. (2005) and Bown et al. (2007): partial msp4 gene was amplified using primers MSP4AP5/MSP4AP3 for the first PCR reaction and msp4f/msp4r for the second reaction. PCR was performed by using 2X PCR Master Mix (Thermo Fisher Scientific, Lithuania). Negative and positive controls were included in all runs. The  nested PCR amplification products of the A. phagocytophilum msp4 gene were extracted from the agarose gel and purified using GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Lithuania) following the  manufacturer’s instructions. The  obtained sequences were analysed using MEGA  X software and compared with the  sequence data available from GenBank using the  BLAST program. The  phylogenetic tree was constructed using the Neighbor-joining method (NJ) with bootstrap analysis of 1000 replicates. The sequences obtained in our study received GenBank accession numbers MT886200–MT886214.

RESULTS AND DISCUSSION

The presence of A. phagocytophilum DNA in the  analysed samples was confirmed by amplification of 381 bp fragments of msp4 gene. A. phagocytophilum DNA was identified in ten (37.04%) of the 27 roe deer. Neither of two examined red deer was infected with A. phagocytophilum. A total of 187 ticks of different developmental stages and sexes were collected from C. capreolus, C. elaphus, and A. alces (Table 1). Nine ticks were identified as Dermacentor reticulatus (four females and five males) and 178 as Ixodes ricinus (89 females, 87 males and two nymphs). For the detection of A. phagocytophilum, each tick was analysed individually. Based on nested PCR assay, A. phagocytophilum DNA was detected in 58.6% (70/178) I. ricinus and 22.2% (2/9) D. reticulatus ticks. The  bacterium was detected only in adult ticks. Both males and females of I. ricinus and D. reticulatus were infected with A. phagocytophilum (Table 1). The incidence of infection varied between different sexes of I.  ricinus: females (48.3%; 43/89) were more frequently infected than males (31.0%; 27/87). Our findings showed that more frequently A. phagocytophilum-infected ticks were found on the roe deer and the moose (Table 1). From one moose 57 ticks of both species (52 I. ricinus and five D. reticulatus) were collected, of which 49.1% (28; 27 I. ricinus and one D. reticulatus) were infected with A.  phagocytophilum. A  total of 105 ticks (104 I.  ricinus, and one D.  reticulatus) were collected from 39 roe deer, and A. phagocytophilum infection was detected in 40.0% (42 I. ricinus ticks) (Table 1). From four red deer, 25 ticks (22 I. ricinus and 3 D. reticulatus) were collected, and A. phagocytophilum DNA was detected in two ticks (one I. ricinus and one D. reticulatus females) (Table 1).

Table 1. Prevalence of Anaplasma phagocytophilum in ticks collected from the cervids in Lithuania

In our previous studies conducted in Lithuania, the  prevalence of A.  phagocytophilum infection in questing D.  reticulatus ticks reached 8% (Paulauskas et al., 2012). However, A. phagocytophilum is found rarely (and at low prevalence) in questing D. reticulatus ticks. In eastern Poland, the prevalence of A. phagocytophilum in questing D. reticulatus varied from 0.7% to 2.0%, depending on the tick collection area (Opalinska et al., 2016; Zajac et al., 2017). The  results obtained in several studies suggest that this tick species is not a  competent vector of A.  phagocytophilum (Zygner  et  al., 2008). The ability of this tick species to transmit A. phagocytophilum has not been yet studied sufficiently. Furthermore, the  presence of pathogens in ticks collected from the host does not provide information whether the source of infection is the  blood of the  host or whether the  pathogen was present in the  tick before feeding (Karbowiaka et al., 2014).

The prevalence of A. phagocytophilum in European I. ricinus tick populations varies. In Lithuania, the prevalence of A. phagocytophilum infection in questing I. ricinus ticks was found to be 2.9% (Paulauskas et al., 2012). In this study, the observed overall prevalence of A. phagocytophilum in I. ricinus feeding on the cervids was high (39.3%). In a similar study conducted in Poland, in total, 238 partially or fully engorged female and 63 non-engorged male I. ricinus ticks were obtained from 51 cervids (20 fallow deer, 18 roe deer, and 13 red deer) (Michalik et al., 2009). The  overall prevalence of A.  phagocytophilum was higher in females (22.7%) than in males (9.1%) of I. ricinus (Michalik et al., 2009). It was noticed that female and male ticks from the PCR-positive cervids were more frequently infected than ticks from the PCR-negative hosts (Michalik et al., 2009). In a study performed in Germany, 331 engorged I. ricinus ticks from 44 roe deer individuals were screened, and 86.1% of them were infected with A.  phagocytophilum (Overzier et al., 2013) with the higher infection rate detected in females (99.0%) than in males (64.8%). It was also noticed that engorged adult ticks removed from the roe deer were significantly more often positive for A.  phagocytophilum than questing adult ticks (Overzier et al., 2013).

Wild ruminants are among the major feeding hosts for ticks in Europe. A great abundance of potential hosts is an important factor for tick expansion and potentially for the  spread of anaplasmosis. There is evidence that higher cervid population densities may influence higher ticks densities, suggesting a positive effect on the prevalence of A. phagocytophilum. A  study conducted in Norway demonstrated that the highest prevalence of A. phagocytophilum in I.  ricinus occurred in locations with the highest densities of the roe deer and the red deer (Rosef et al., 2009). In Europe, A. phagocytophilum has been detected in local wild ruminant species with different prevalence (reviewed in Stuen et al., 2013). The roe deer show A.  phagocytophilum prevalence rates reaching up to 98.9%, and several genetic variants of A.  phagocytophilum (both potentially pathogenic and non-pathogenic) have been found in the roe deer in Europe (Overzier et al., 2013). It has been suggested that the roe deer mainly acts as a reservoir of several A. phagocytophilum non-pathogenic variants for other animal species (Stuen et al., 2013). Other cervid species in Europe may also constitute efficient reservoir hosts of A. phagocytophilum as the pathogen has been detected in the red deer with up to 87% prevalence, in the  fallow deer (Dama dama) with up to 72%, in the sika deer (Cervus nippon) with up to 50%, and in the moose with up to 42.9% (reviewed in Stuen et al., 2013; Pūraitė et al., 2015; Ražanskė et al., 2019).

The  sequence analysis of the  msp4 gene among the  15 samples derived from the  roe deer (n = 8) and I. ricinus (n = 6) and D. reticulatus (n  =  1) ticks revealed ten different sequence types: five msp4 sequence types were detected in ticks and seven in roe deer. Two sequence variants (1 and 2) were detected in both I.  ricinus ticks and roe deer (Table  2). The analysed sequences showed 98–100% homology to each other (differing at one to 32 nucleotide positions) and to the other msp4 gene sequences deposited in GenBank. Ambiguous nucleotides were observed in msp4 sequences of A. phagocytophilum derived from four roe deer (variants 4, 5, 7). In all cases, ambiguous nucleotides were detected at positions where in other sequences either one of the two possible nucleotides were found, possibly indicating double infections (Table 2). Four A. phagocytophilum sequences (variant  1) derived from the  roe deer (sample MT886207) and from I. ricinus ticks obtained from one moose (sample MT886200) and two roe deer individuals (samples MT886201 and MT886203) were 100% identical to each other and to other European sequences reported from I.  ricinus (Slovenia, KM205439) and D. reticulatus (Lithuania, JN181091) ticks and the roe deer from Germany (KU712165) and Slovenia (KM205437) (Figure). Four A. phagocytophilum msp4 sequence variants that were found in five roe deer (samples MT886214, MT886208, MT886209, MT886210, MT886212) had unique nucleotide composition (variants 5, 6, 7, 10) and therefore differed from other previously identified A. phagocytophilum sequences in the GenBank database (Table 2). A. phagocytophilum msp4 sequence derived from D. re-ticulatus tick (sample MT886202) collected from the  red deer (variant  9) differed from other msp4 sequences detected in this study and was identical to the  sequences derived from the  roe deer in Slovakia (EU180060) and Hungary (MF974860) (Table  2, Figure). One roe deer (sample MT886212) harboured A.  phagocytophilum msp4 sequence (variant 10), which markedly differed from other sequences detected in ticks and roe deer in this study. The closely related A. phagocytophilum msp4 sequence (differed at one nucleotide position) was identified in the roe deer in Poland (Figure).

Table 2. Differences in the msp4 nucleotide sequences among A. phagocytophilum strains detected in ticks and roe deer in Lithuania

The majority of the  A.  phagocytophilum strains circulating in Europe are associated with the  infection of the  ruminants (Stuen  et  al., 2013). Previous studies demonstrated high sequence heterogeneity among A. phagocytophilum strains in msp4 gene isolated from different ruminant species. Bown et al. (2007) reported high variability of A.  phagocytophilum msp4 sequences (11  variants) derived from 20 different hosts from several European countries and the  USA. Six different A.  phagocytophilum msp4 gene variants have been identified in the roe deer and six in the red deer in Norway (Ražanskė et al., 2019). In previous studies conducted in Lithuania, seven msp4 gene variants of A. phagocytophilum were detected in I. ricinus and Dermacentor reticulatus ticks (Paulauskas  et  al., 2012). Most of A.  phagocytophilum strains detected in I. ricinus and D. reticulatus ticks collected in Lithuania are identical to the strains derived from various cervid species (Paulauskas et al., 2012).

CONCLUSIONS

Our results provide new information on the prevalence and distribution of A. phagocytophilum strains in the roe deer and I. ricinus and D. reticulatus ticks collected from the roe deer, the red deer, and the moose in Lithuania. The  results of the  present study demonstrate high infection rates of A.  phagocytophilum in ticks parasitizing different cervid species and the  possible risk of transmitting these infections to their hosts. Five msp4 gene variants of A. phagocytophilum were detected in ticks and seven in red deer. Ten A. phagocytophilum msp4 sequences derived from ticks and the roe deer in this study had been previously described in ticks, the roe deer, the red deer, and the moose in other European countries, while five sequences derived from the roe deer differed from other previously identified A. phagocytophilum msp4 gene sequences in the GenBank database.

References

Jana Radzijevskaja, Justina Snegiriovaitė, Artūras Kibiša, Irma Ražanskė, Algimantas Paulauskas

ANAPLASMA PHAGOCYTOPHILUM INFEKCIJOS, NUSTATYTOS LIETUVOJE ELNINIAMS GYVŪNAMS IR JUOS PARAZITUOJANČIOMS ERKĖMS, MOLEKULINĖ CHARAKTERISTIKA