Abstract
Anaplasma phagocytophilum is the causative agent of granulocytic anaplasmosis in humans, dogs, cats, horses and tick-borne fever in ruminants. In Europe, its main vector is the tick species Ixodes ricinus. In this study, spleen and liver samples, as well as ticks from 18 wild-living mammals (belonging to seven species) were analysed for the presence of A. phagocytophilum with molecular methods. The zoonotic ecotype-I of A. phagocytophilum was identified in a European wildcat (Felis silvestris) and its tick, a European pine marten (Martes martes) and a Eurasian red squirrel (Sciurus vulgaris). All PCR-positive samples were collected in 2019 and originated in the same geographic area. These results indicate that taxonomically diverse mammalian species can maintain the local enzootic cycle of the same genotype of A. phagocytophilum. To the best of our knowledge, this is the first report of the zoonotic variant of A. phagocytophilum in the wildcat and in the European pine marten in a broad geographical context, as well as in the red squirrel in Hungary. Since all these host species are well known for their urban and peri-urban presence, the results of this study verify their role in the synanthropic enzootic cycle of granulocytic anaplasmosis and tick-borne fever.
Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) is a tick-borne, Gram-negative bacterium species with a broad geographical distribution in the northern hemisphere (Stuen et al., 2013a). It develops in neutrophils, thus causing a disease known as granulocytic anaplasmosis in humans, dogs, cats, horses and tick-borne fever in ruminants (Stuen et al., 2013a). Based on serological and molecular evidence from countries in the geographical region of Hungary (Central and Southeastern Europe), co-infections of horses and dogs with other tick-borne pathogens and A. phagocytophilum might enhance the pathogenic effect of the latter (Mircean et al., 2012; Krämer et al., 2014; Huber et al., 2017; Kovačević Filipović et al., 2018; Tsachev et al., 2019; Drážovská et al., 2021).
In Europe, the main vector of A. phagocytophilum is Ixodes ricinus (Woldehiwet, 2010). This is a generalist, three-host tick species that may feed on a broad range of reservoirs of A. phagocytophilum, and thus may overbridge wild and domestic animals as well as humans, implying the risk of zoonotic spread. In the absence of its transovarial transmission by female ticks, A. phagocytophilum cannot survive across several tick generations, and the maintenance of its natural and urban enzootic cycles is based on transstadial transmission via successive tick developmental stages (Jaarsma et al., 2019). This necessitates the alternating participation of tick vectors and vertebrate hosts in the development of A. phagocytophilum, increasing the spectrum of evolutionary pressures and thus its genetic variation.
Based on its transmission dynamics, hosts, ecological and genetic properties, A. phagocytophilum was shown to belong to four major ecotypes, among which ecotype-I has zoonotic potential (Jahfari et al., 2014). This zoonotic ecotype has the broadest range of wildlife reservoirs but may also infect certain domestic animals (Jahfari et al., 2014). In particular, the most important synanthropic hosts of ecotype-I are dogs, cats, horses, and several wild living urban or peri-urban mammals, as exemplified by wild boars, red foxes and hedgehogs, but not birds and murine or cricetid rodents (Jahfari et al., 2014; Matei et al., 2019; Jaarsma et al., 2019). However, the epidemiological role of non-canid wild carnivores and non-murine/cricetid rodents remains to be elucidated, as relevant data were published only sporadically and the ecotype(s) these hosts may harbour is (are) not always reported (e.g., in Matei et al., 2021).
In Hungary, infection with A. phagocytophilum was reported in various hosts, including small mammals (Rigó et al., 2011), dogs (Hornok et al., 2013), hedgehogs (Földvári et al., 2014), birds (Hornok et al., 2014a), red foxes (Tolnai et al., 2015) and large game animals (Hornok et al., 2018a), as well as in the main tick vector, Ixodes ricinus (Sréter et al., 2004; Egyed et al., 2012; Hornok et al., 2014b). However, while this tick species is known to occur on wild felids, mustelids and sciurid rodents in the country (Hornok et al., 2020, 2022a), there are no data on the A. phagocytophilum infection status of these mammals, like in several other regions of Central and Eastern Europe. The aim of this study was to compensate for this lack of epidemiological data.
Spleen and liver samples were collected from seven species of wild mammals, including European wildcats (Felidae: Felis silvestris, n = 4); one raccoon dog (Canidae: Nyctereutes procyonoides); four species of Mustelidae: beech martens (Martes foina, n = 3), European pine martens (Martes martes, n = 3), least weasels (Mustela nivalis, n = 2) and one Eurasian otter (Lutra lutra); as well as Eurasian red squirrels (Sciurus vulgaris, n = 4). These animals were found dead due to natural causes or as road-kills, between 2015 and 2021, in northeastern Hungary, in the Aggtelek National Park and its surroundings. This region (48° 30′ N, 20° 36′ E) is a low, karstic area with altitudes of 150–604 m covered by various deciduous forests (mainly oak and hornbeam). The landscape is dominated by systems of karstic plateaus dissected by deep valleys. The climate is humid continental with long summers. The average annual temperature is rather low, 8.2 °C, which figure is typical only of higher elevations in Hungary. The annual precipitation was formerly between 600–700 mm but it significantly decreased in the last few years to about 400–500 mm. Biogeographically this region displays a mosaic-like transition between the higher mountains of the Carpathians and the lowlands of the Pannonian Basin (Varga, 1999). From a biogeographical point of view, the study area is part of the Pannonic region. The sampled species are widespread and regularly observed in the area. However, no specific population surveys have been carried out so far, so the exact size of the populations is currently unknown.
The fur covering of each animal was carefully checked for the presence of ticks which were collected into and stored in 96% ethanol. Tick species were identified according to standard keys (Estrada-Peña et al., 2017). The DNA extracts of ticks originating from A. phagocytophilum PCR-positive hosts were also examined in this study.
DNA was extracted from host tissue samples and from whole ticks (individually) with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction, including an overnight digestion in tissue lysis buffer and proteinase-K at 56 °C in case of the latter, as reported by Hornok et al. (2014b, 2018a). An extraction control (tissue lysis buffer) was also processed in each set of samples.
In the screening assay, the primers EHR16SD (5′-GGT ACC YAC AGA AGA AGT CC-30) and EHR16SR (5′-TAG CAC TCA TCG TTT ACA GC- 3′) were used, which amplify an approximately 350-bp-long fragment of the 16S rRNA gene from various members of Anaplasmataceae (Brown et al., 2001), modified as reported (Hornok et al., 2018b). From samples positive in the screening PCR, amplification of an approx. 600-bp-long fragment of the heat shock chaperonin (GroEL) gene of A. phagocytophilum was also attempted (Alberti et al., 2005). The primers EphplGroEL(569)F (5′-ATG GTA TGC AGT TTG ATC GC-3′) and EphGroEL(1142)R (5′-TTG AGT ACA GCA ACA CCA CCG GAA-3′) were used as reported (Hornok et al., 2022b). Sequence-verified A. phagocytophilum DNA from a dog (code VE39) was used as positive control.
Purification and sequencing of the PCR products were done by Biomi Ltd. (Gödöllő, Hungary). Obtained sequences were manually edited, then aligned with GenBank sequences by nucleotide BLASTN program (https://blast.ncbi.nlm.nih.gov). Four species-specific sequences were submitted to GenBank (A. phagocytophilum GroEL: ON186490-ON186493).
The results are summarised in Fig. 1. The spleen and liver samples of two carnivores, a European wildcat (F. silvestris) and a European pine marten (M. martes), and only the spleen sample (but not the liver DNA extract) of a Eurasian red squirrel (Sciurus vulgaris) were positive in the 16S rRNA gene PCR to detect Anaplasmataceae. This is in line with the observation that the spleen is a more likely source of A. phagocytophilum detection than the liver (Matei et al., 2021).
Summary of results from molecular analyses of Anaplasma phagocytophilum-infected hosts. The latter included one of four European wildcats (Felis silvestris), one of three European pine martens (Martes martes) and one of four Eurasian red squirrels (Sciurus vulgaris). In the first row, these host species are shown with their silhouettes in this order, respectively. Abbreviations: F – female, N – nymph, L – larva; NA – not available
Citation: Acta Veterinaria Hungarica 70, 3; 10.1556/004.2022.00021
Summary of results from molecular analyses of Anaplasma phagocytophilum-infected hosts. The latter included one of four European wildcats (Felis silvestris), one of three European pine martens (Martes martes) and one of four Eurasian red squirrels (Sciurus vulgaris). In the first row, these host species are shown with their silhouettes in this order, respectively. Abbreviations: F – female, N – nymph, L – larva; NA – not available
Citation: Acta Veterinaria Hungarica 70, 3; 10.1556/004.2022.00021
Summary of results from molecular analyses of Anaplasma phagocytophilum-infected hosts. The latter included one of four European wildcats (Felis silvestris), one of three European pine martens (Martes martes) and one of four Eurasian red squirrels (Sciurus vulgaris). In the first row, these host species are shown with their silhouettes in this order, respectively. Abbreviations: F – female, N – nymph, L – larva; NA – not available
Citation: Acta Veterinaria Hungarica 70, 3; 10.1556/004.2022.00021
Amplification of the heat shock protein (GroEL) gene from these samples yielded identical sequences, with 100% (517/517 bp) identity to several sequences of ecotype-I of A. phagocytophilum deposited in GenBank (e.g., MN093180 from I. ricinus, The Netherlands: Jaarsma et al., 2019; MW366836 from wild boar-infesting ticks, Hungary: Hornok et al., 2022b). Interestingly, all PCR-positive samples were collected in the same year, 2019 and originated in the same geographic area around Szalonna (co-ordinates: 48.45093°N 20.74045°E). This means that sympatric, taxonomically diverse mammals, a wildcat, a pine marten and a red squirrel were infected with the same GroEL variant of A. phagocytophilum. Unlike this genetic consistency between three mammalian species from different orders and families, among sympatric ruminants, i.e., red deer and sheep (which both tend to harbour ecotype-I of A. phagocytophilum: Jahfari et al., 2014) identical msp4 genotypes were demonstrated only among individuals of the same host species (Stuen et al., 2013b). While it was reported that A. phagocytophilum ecotype-I has a broad host spectrum on a continental scale (Jaarsma et al., 2019), the results of this study prove that diverse mammalian species can maintain the local enzootic cycle of the same genotype.
Considering ticks collected from the three A. phagocytophilum PCR-positive mammals, one of the two I. ricinus females collected from the wildcat also harboured this GroEL variant. This may reflect that either the source of A. phagocytophilum-infection in this wildcat was the PCR-positive tick collected from it, or the tick ingested blood during bacteraemia of its host. It might be relevant to note in the context of both possibilities that the probability of transstadial transmission of A. phagocytophilum appears to be stage dependent: based on prevalence rates after acquisition feeding, it is more likely that adults of I. ricinus will harbour (and thus inoculate) A. phagocytophilum than nymphs (Ogden et al., 2003). Unlike in the case of the wildcat, all 18 ticks (belonging to four species: Fig. 1) collected from the pine marten were PCR negative. The most likely explanation for this is that the pine marten with PCR-positive spleen and liver samples was not in the state of bacteraemia. This is supported by the fact that none of the ticks collected from this host in different states of engorgement were PCR positive, and A. phagocytophilum is known to cause waving bacteraemia (Granquist et al., 2010). On the other hand, no ticks were found on the PCR-positive red squirrel.
In summary, although the sample size was relatively small in this study and all seven mammalian species involved were represented by only one to four individuals, the results are new in an international context. First, to the best of our knowledge, this is the first report of the zoonotic variant of A. phagocytophilum in wildcat in a worldwide context, because the ecotype of this pathogen reported recently in the same host species was not identified (Matei et al., 2021). Similarly, while a broad range of mustelids were screened for A. phagocytophilum in Western Europe (Hofmeester et al., 2018), ecotype-I could only be identified in the European polecat (Mustela putorius) but not in European pine martens as shown here for the first time. In addition, the Eurasian red squirrel was reported to harbour both ecotypes I and II in Western Europe (Belgium: Ruyts et al., 2017), ecotypes I, II and IV in Central Europe (Czech Republic: Lesiczka et al., 2021) and here it was identified for the first time as the host of ecotype-I in Hungary. Since all three host species of the zoonotic ecotype of A. phagocytophilum are well known for their urban or peri-urban presence (Fingland et al., 2021; Urzi et al., 2021; Wereszczuk et al., 2021), these results verify their role in the synanthropic enzootic cycle and epidemiology of granulocytic anaplasmosis and tick-borne fever.
Acknowledgement
This study was funded by Project no. TKP2020-NKA-01 implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the ‘Tématerületi Kiválósági Program 2020’ (2020-4.1.1-TKP2020) funding scheme.
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