Rickettsial infection in ticks infesting wild birds from two eco‑regions of Argentina

Several tick-borne Rickettsia species are recognized human pathogens in Argentina. Here we evaluated rickettsial infection in ticks collected on passerine birds during 2011-2012 in two eco-regions of Argentina. The ticks were processed by molecular analysis through polymerase chain reaction (PCR) detection and DNA sequencing of fragments of two rickettsial genes, gltA and ompA. A total of 594 tick specimens (532 larvae and 62 nymphs), representing at least 4 species ( Amblyomma tigrinum, Ixodes pararicinus, Haemaphysalis juxtakochi, Haemaphysalis leporispalustris ), were evaluated. At least one A. tigrinum larva, collected on Coryphospingus cucullatus in Chaco Seco, was infected with Rickettsia parkeri, whereas at least 12 larvae and 1 nymph of I. pararicinus, collected from Troglodytes aedon, Turdus amaurochalinus, Turdus rufiventris, C. cucullatus and Zonotrichia capensis, were infected with an undescribed Rickettsia agent, genetically related to several rickettsial endosymbionts of ticks of the Ixodes ricinus complex. R. parkeri is a recognized human pathogen in several American countries including Argentina, where a recent study incriminated A. tigrinum as the potential vector of R. parkeri to humans. Birds could play an important role in dispersing R. parkeri- infected A. tigrinum ticks. Additionally, we report for the first time a rickettsial agent infecting I. pararicinus ticks.

Wild birds can be hosts of different stages of some species of ticks, commonly larvae and nymphs, and rarely adults (GUGLIELMONE et al., 2014;FLORES et al., 2014). Furthermore, wild birds are among the most mobile hosts, and therefore they may be regarded as hosts with relevant potential in the dispersion of ticks and tick-borne diseases, including rickettsial organisms (ELFVING et al., 2010;HORNOK et al., 2014;BERTHOVÁ et al., 2016). Among 127 species of Ixodidae established in the Neotropical Zoogeographic Region (GUGLIELMONE et al., 2014;NAVA et al., 2014a, b;KRAWCZAK et al., 2015), 39 are found in Argentina (GUGLIELMONE & NAVA, 2005, 2006NAVA et al., 2009NAVA et al., , 2014a. Most of these species belong to the genus Amblyomma, best represented in Argentina with 25 species. Ten species from three genera have been reported parasitizing wild birds in different Argentinean eco-regions (FLORES et al., 2014); however, bird ticks have never been searched for rickettsial infection in Argentina. Here, we have evaluated rickettsial infection in ticks collected on wild birds in two eco-regions of Argentina.
This study was conducted in two localities of the Chaco Seco eco-region (Chaco Seco1: 30°50'S, 62°54'W; and Chaco Seco 2: 30°22'S, 64°21'W) and one in the Yungas ecoregion (Parque Nacional El Rey: 24°43'S, 64°38'W) in Argentina as defined by Burkart et al. (1999). Bird collections were performed using mist nets, which remained active during morning and twilight hours, obtaining convenience samplings in 2011 and 2012, as previously reported (FLORES et al., 2014). Each individual bird was identified using Narosky & Yzurieta (2010), classified under Clements et al. (2015) criteria and examined for ticks using fine-tipped tweezers. After being processed, the birds were released, and the ticks obtained were stored in 70% ethanol until specific determination in the laboratory. The detailed procedures for the fieldwork, bird and tick taxonomic identification, and the results of the ticks infesting these birds have been published elsewhere (FLORES et al., 2014).
Ticks of the same developmental stage, collected from the same individual host, were processed in pools of 1 to 39 larvae (median: 4) or 1 to 4 nymphs (median: 1). Ticks from each pool were crushed with a sterile pestle in a microtube, and then processed for DNA extraction by using the AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Axygen Biosciences, USA) following the manufacturer's procedures. DNA samples were processed by PCR using primers CS-78 and CS-323 targeting a 398-bp fragment of the citrate synthase gene (gltA), which occurs in all Rickettsia species (LABRUNA et al., 2004). Samples positive by this first PCR assay were tested by a second PCR assay with primers Rr190.70p and Rr190.701, which amplifies a ≈630-bp fragment of the 190-kDa outer membrane protein gene (ompA) of the majority of the SFG Rickettsia species (ROUX et al., 1996). PCR products were DNA-sequenced and subjected to BLAST analyses (BLAST, 2015) to infer the closest similarities available in GenBank.
A total of 594 tick specimens (532 larvae and 62 nymphs), representing at least 4 species, were evaluated in 124 pools for rickettsial infection (Table 1). A complete list of hosts for these ticks has been published elsewhere (FLORES et al., 2014). Two tick species were found to be infected by Rickettsia. One Amblyomma tigrinum pool of 4 larvae was infected with R. parkeri, as its gltA and ompA DNA partial sequences (308 bp and 551 bp, respectively) were 100% identical to R. parkeri strain Maculatum20 (GenBank accession numbers U59732 and U43802, respectively).
Thirteen pools of Ixodes pararicinus (12 out of 32 larval pools; and 1 out of 8 nymphal pools) yielded PCR products for both the gltA and the ompA genes. DNA sequences were successfully obtained for 5 pools, which generated identical sequences for each rickettsial gene. The GenBank nucleotide sequence accession numbers for the partial sequences generated in this study are KU744411-KU744412 for the gltA and ompA genes of R. parkeri from A. tigrinum and KU744413-KU744414 for the gltA and ompA genes of Rickettsia sp. from I. pararicinus.
This study provides molecular evidence of two rickettsial agents infecting ticks that were parasitizing birds in Argentina. R. parkeri is a recognized human pathogen in several countries of the Americas (PAROLA et al., 2013), including Argentina, where a recent study incriminated A. tigrinum as a probable vector of R. parkeri to humans (ROMER et al., 2014). The tick A. tigrinum is known to occur in a variety of eco-regions among almost all South American countries (ESTRADA-PEÑA et al., 2005). Recent findings of R. parkeri infecting A. tigrinum adult ticks in Bolivia (TOMASSONE et al., 2010), Uruguay (LADO et al., 2014), and Argentina (ROMER et al., 2014) suggest that this pathogen could have a wide distribution within the South American population of this tick species. Thus, it is likely that wild birds could play an important role in dispersing R. parkeri-infected A. tigrinum ticks, which could expand the area of human exposure to these ticks, consequently increasing the risk of human rickettsiosis. On the other hand, because all tick-infested birds of this study are considered to be non-migratory species (NAROSKY & YZURIETA, 2010), their specific role in dispersing infected ticks over long distances should be limited.
Additionally, we report for the first time a rickettsial agent infecting I. pararicinus ticks. Partial molecular characterization of this agent indicates that it is most closely related to SFG rickettsial agents associated with I. affinis (LOPES et al., 2016), I. ricinus (MAIOLI et al., 2012), I. minor (OGRZEWALSKA et al., 2015), and I. scapularis (PAROLA et al., 2013). These four Ixodes species, as well as I. pararicinus, belong to the I. ricinus species complex, based primarily on morphological and genetic relatedness (KEIRANS et al., 1985;BARBIERI et al., 2013). Interestingly, recent studies have indicated that tick members of the I. ricinus species complex are usually infected by species-specific closely related rickettsial organisms, usually considered endosymbionts (KURTTI et al., 2015). Moreover, the I. ricinus associated R. monacensis is considered by some authors as a human pathogen (PAROLA et al., 2013). Further studies on isolation and deeper molecular characterization are needed to elucidate the taxonomic status of the rickettsial endosymbiont of I. pararicinus.