Detection of Anaplasma phagocytophilum, Candidatus Neoehrlichia sp., Coxiella burnetii and Rickettsia spp. in questing ticks from a recreational park, Portugal

Abstract

Tick-borne agents with medical relevance have been recorded in Portugal but little is known about their occurrence in urban outdoor leisure areas. This study aimed to investigate ticks and tick-borne agents in three public parks of Lisbon’s metropolitan area. A total of 234 questing ticks belonging to eight species were found in Parque Florestal de Monsanto (PFM). Ixodes ventalloi represented 40% of collections. Mitochondrial genes confirmed Ixodes morphological identification, evidencing the intraspecific variability of I. ricinus and particularly I. frontalis populations. Regarding tick-borne agents, Rickettsia massiliae DNA were found in 21 (9.0%) ticks, Coxiella burnetii in 15 (6.4%), Anaplasma phagocytophilum in five (2.1%), an agent closely related to Candidatus Neoehrlichia mikurensis in two (0.9%), Rickettsia sibirica mongolitimonae and Rickettsia monacensis each in one (0.4%). Active enzootic cycles were suggested for these agents by the detection of positives in different time periods. Five tick species were founded with C. burnetii, including I. ventalloi which seems to be a new association record. This tick was also the only species found positive for A. phagocytophilum and the Candidatus Neoehrlichia mikurensis-like agent. Two A. phagocytophilum variants were detected in PFM, one of them representing a potentially new ecotype already found in I. ventalloi from another Portuguese area. To the authors´ knowledge, this is also the first report of such a Candidatus Neoehrlichia mikurensis-like microorganism. These data show an interesting diversity of ticks and tick-borne agents with potential public health relevance in PFM, an urban recreational area commonly frequented by humans and their pets.

Introduction

Green spaces in cities are regarded as most beneficial for urban environment and population well-being by contributing to improve air quality, reduce soil erosion and regulate climate, and by creating important recreational areas. These habitats can also take part in conservation strategies and promotion of biodiversity. Urban and peri-urban forest parks and large city gardens can support a variety of vertebrates, including resident and migratory birds, lizards, rodents and other small to medium sized mammals (Rizzoli et al., 2014; Uspensky, 2014). The occurrence of such animal populations, sometimes along with free-roaming domestic species, is also linked with the flourishment of invertebrate and microbiological communities of parasites and pathogens, some with medical implications as ticks and tick-borne pathogens (Comer et al., 2001; Jahfari et al., 2017). Thus, green spaces in cities may represent a unique interface where humans and their pets can regularly be exposed to ticks (Dobson et al., 2011; Jennett et al., 2013). Several tick-borne pathogens are actually becoming increasingly recognized in urban and sub-urban public recreational areas (Overzier et al., 2013; Didyk et al., 2017; Hansford et al., 2017).

Bacteria belonging to the order Rickettsiales are among the most important tick-borne agents with medical implications in southern Europe. Their relevance resides in the variety of species recorded, including agents traditionally implicated in disease, emergent or newly recognized human pathogens, and also in their potentially expanding geographic range. The etiological agent of Mediterranean spotted fever, Rickettsia conorii Malish strain is one of the best examples. It is largely diagnosed in Mediterranean countries but sporadic cases are also reported in non-endemic areas of central and northern Europe due to vector introduction or imported cases in travellers returning from endemic areas (Koenen et al., 2013). Other spotted fever group Rickettsia of medical relevance include R. conorii Israeli strain, R. sibirica mongolitimonae (Lymphangitis-associated rickettsiosis, LAR) and R. slovaca (Tick-borne lymphadenopathy, TIBOLA or Dermacentor-borne necrosis erythema and lymphadenopathy, DEBONEL) (Raoult et al., 1996; Raoult and Roux, 1997; Bacellar et al., 1999; Giammanco et al., 2005; De Sousa et al., 2006, 2013; Koenen et al., 2013). A potential medical role is also suggested for several other broadly distributed rickettsiae in Europe such as R. helvetica, R. massiliae and R. monacensis (Nilsson et al., 1999; Vitale et al., 2006; Jado et al., 2007). The order Rickettsiales also includes Anaplasma, a genus traditionally linked to veterinary diseases but containing at least one zoonotic species, A. phagocytophilum. This agent is implicated in human granulocytic anaplasmosis, one of the most reported tick-borne disease in the eastern USA (Dumler et al., 2007). So far, the disease is less prevalent in Europe and mostly associated with central European areas despite the broad geographical distribution of A. phagocytophilum, possibly due to the circulation of less pathogenic strains or to an under-evaluated situation (Stuen et al., 2013; Jahfari et al., 2014). Candidatus Neoehrlichia mikurensis, a potential new taxon of Rickettsiales, represents another increasingly recognized tick-borne agent with human health implications (von Loewenich et al., 2010; Welinder-Olsson et al., 2010). Moreover, there are other pathogens considered secondarily vectored by ticks but intimately linked to these arthropods, such as Coxiella burnetii. This Legionellales member is an air-borne agent endemic in Europe, implicated in Q fever sporadic cases and outbreaks (Maurin and Raoult, 1999). The latest and the largest ever reported Q fever outbreak started in 2007 in the south of the Netherlands (Schimmer et al., 2008). Despite its air-borne nature, the agent seems to have evolved from a tick-endosymbiont lineage (Duron et al., 2015). Indeed, classic studies recorded C. burnetii in several tick species and although their role in human infection seems negligible, they are regarded as relevant for the maintenance of C. burnetii sylvatic cycles (Maurin and Raoult, 1999; Sprong et al., 2012).

Presently, four of the above-mentioned agents are linked to human disease in Portugal. The medical relevance has been shown for a long time for R. conorii (Malish and Israeli strain), C. burnetii and more recently also confirmed for R. sibirica mongolitimonae and R. slovaca (De Sousa et al., 2006, 2013; Koenen et al., 2013; Santos, 2017). Except for Candidatus Neoehrlichia mikurensis, all the above mentioned agents have also been identified in ticks across the country (Santos-Silva et al., 2011, 2016). Despite the diversity and broad distribution of Rickettsiales and C. burnetii in Portugal, the occurrence of these agents’ enzootic cycles in urban centers is still poorly investigated. The present work aims to characterize the tick fauna and to investigate the circulation of Spotted fever group (SFG) Rickettsia, A. phagocytophilum, Candidatus Neoehrlichia mikurensis and C. burnetii in public parks located in Lisbon’s metropolitan area.