Anaplasma platys and Rickettsia massiliae in Rhipicephalus sanguineus sensu stricto ticks collected on dogs in the Patagonian region of Argentina

Abstract

The aim of this study was to examine the presence of tick-borne rickettsial bacteria in Rhipicephalus sanguineus sensu stricto ticks collected from dogs in the Patagonian region of Argentina. Fourteen stray dogs from Valcheta, Río Negro province, Argentina were examined for the presence of R. sanguineus s.s. ticks. Ninety ticks were collected and identified to species level. DNA was extracted and analysed by conventional PCR assays for the presence of tick-borne bacteria belonging to the genera Anaplasma, Ehrlichia and Rickettsia. Thirty-three tick pools were tested by different PCR assays of which 3 were positive for Anaplasmataceae bacteria. From the 3 Anaplasmataceae positive samples, 2 partial 16S rDNA sequences were generated and belonging to Anaplasma platys, the causative agent of canine cyclic thrombocytopenia. Two tick samples were positive in Rickettsia specific PCR assays and were identified by phylogenetic analysis as Rickettsia massiliae, a member of the spotted fever group rickettsiae. The results of this study demonstrate the molecular detection of 2 rickettsial bacteria in R. sanguineus s.s. in a region of Argentina where no data were available so far.

Introduction

The order Rickettsiales includes obligate intracellular alpha proteobacteria and contains the families of Anaplasmataceae, Midichloriaceae and Rickettsiaceae (Ferla et al., 2013). Within this order, several species are of particular importance for veterinary and public health. Anaplasma platys and Ehrlichia canis, the causative agents of canine cyclic thrombocytopenia and canine monocytic ehrlichiosis, respectively, are amongst the most important bacteria in veterinary medicine (Harrus et al., 2012; Atif et al., 2021; Diniz and Moura de Aguiar, 2022). Rickettsia massiliae, a member of the spotted fever group rickettsiae, is known to be zoonotic human cases are reported (García-García et al., 2010; Oteo and Portillo, 2012; Portillo et al., 2015).

The main vector of these bacteria is ticks from the Rhipicephalus sanguineus group (Bremer et al., 2005; Parola et al., 2008). In Argentina, 2 different taxa, namely R. sanguineus sensu stricto (previously known as R. sanguineus sensu lato ‘temperate lineage’) and R. linnaei (previously known as R. sanguineus sensu lato ‘tropical lineage’) (Nava et al., 2017, 2018; Šlapeta et al., 2022) are present. Rhipicephalus linnaei was found only in the provinces of Formosa and Salta, above 24° southern latitude, while R. sanguineus sensu stricto is distributed in almost all regions of Argentina (Nava et al., 2012; Cicuttin et al., 2015a, 2015b; Sebastian et al., 2021; Copa et al., 2023). Besides A. platys, E. canis and R. massiliae, ticks from the R. sanguineus group are involved as potential vector of other pathogenic agents such as Babesia vogeli, Babesia gibsoni and Hepatozoon canis (Dantas-Torres and Figueredo, 2006; Baneth and Allen, 2022).

Knowledge of tick-borne microorganism in the Patagonian region of Argentina is scarce. Borrelia burgdorferi sensu lato was detected in Ixodes sp. cf. I. neuquenensis and I. sigelos collected from rodents in the provinces of Chubut, Río Negro and Santa Cruz (Sebastian et al., 2016), Hepatozoon sp. in Amblyomma tigrinum collected on foxes in Santa Cruz province (Millán et al., 2018) and ‘Candidatus Rickettsia andeanae’ and Ehrlichia sp. in Amblyomma pseudoconcolor collected on Chaetophractus villosus in Río Negro province (Sebastian et al., 2022). Further, Winter et al. (2024) reported the molecular detection of ‘Candidatus Anaplasma boleense’ and two strains of Ehrlichia sp. in A. tigrinum collected on different hosts and of the vegetation from Rio Negro province, within Ehrlichia sp. strain Viedma which may be present in this geographical region associated with the tick species A. pseudoconcolor and A. tigrinum. However, there are no studies on ticks from the R. sanguineus group in the Patagonian region of Argentina.

Therefore, the aim of the study was to investigate the presence of tick-borne rickettsial bacteria with veterinary and public health relevance in R. sanguineus s.s. collected on dogs in the Patagonian region from Argentina.

Material and methods

Sampling site, tick sampling and identification

Between November 2022 and August 2023, ticks were collected on 14 stray dogs in Valcheta (40°40′47.5″S; 66°09′57.3″W), Río Negro province, Patagonian region, Argentina. This small village is located in a transition area between the ‘Monte’ and ‘Estepa’ phytogeographic areas (Oyarzabal et al., 2018).

Ticks were collected manually from the dogs during routine veterinary treatment and investigation. Afterwards, the collected ticks were stored in ethanol (96%), and morphologically determined following Nava et al. (2018).

Molecular detection of tick-borne rickettsial bacteria

Before DNA extraction, ticks were successively washed in hypochlorite (10 vol%), PBS (1X) and distilled water. Afterwards, ticks were pooled in batches of 1–10 specimens, according to tick development stage and individual host. The DNA extraction was carried out with the High Pure PCR Template Preparation Kit (Roche, Germany) according to the manufacturer's instructions. Concentration and purity of the DNA samples were verified via Nanodrop measurements. For the detection of rickettsial bacteria, 4 conventional PCR assays were carried out. Shortly, 2 assays detecting different fragments of the 16S rDNA gene of Anaplasmataceae family members were used for the detection of Anaplasma spp. and Ehrlichia spp. while the genes gltA and ompA served as targets for the detection of Rickettsia species. Used oligonucleotides and assay references are given in Table 1. In all PCR reactions, ultra-pure water was used as negative control while DNA of Anaplasma marginale and Rickettsia parkeri were chosen as positive control for the detection of the genes for 16S rRNA and gltA/ompA, respectively.

Table 1. Oligonucleotides used for the molecular detection of rickettsial bacteria in DNA samples of Rhipicephalus sanguineus sensu stricto ticks collected on dogs from Valcheta, Rio Negro province, Patagonia, Argentina

Data analysis

Positive PCR amplicons were purified using the High Pure PCR Product Purification Kit (Roche, Germany) and sent to INTA Castelar (Genomics Unit, Buenos Aires, Argentina) for sequencing. Obtained partial sequences were edited using BioEdit Sequence Alignment Editor (Hall, 1999) with optional manual edition, aligned with the program Clustal W (Thompson et al., 1994), and compared with sequences deposited in GenBank. Phylogenetic analyses for the gltA and ompA partial sequences were performed with maximum-likelihood (ML) methods by using the program Mega X (Kumar et al., 2018). Best-fitting substitution models were determined with the Akaike Information Criterion using the ML model test implemented in MEGA X. Support for the topologies was tested by bootstrapping over 1000 replications, and gaps were excluded from the comparisons.

Results and discussion

A total of 90 specimens of R. sanguineus s.s. ticks (3 larvae, 72 nymphs, 6 females and 9 males) were collected on 14 dogs from Valcheta, Río Negro province (see Fig. 1, Table 2). Additionally, a co-infestation of R. sanguineus s.s. ticks with 1 female of A. tigrinum was observed (this tick was not included in the rickettsial bacterial detection assay). Both tick species are known to infest dogs and were also previously identified in the Patagonian region of Argentina (Nava et al., 2017).

Figure 1. Map of Rio Negro province, Patagonia, Argentina showing the sample locality (Valcheta: 40°40′47.5′′S; 66°09′57.3′′W) of the Rhipicephalus sanguineus sensu stricto ticks used for the detection of tick-borne rickettsial bacteria. The map was created with QGIS version Prizren 3.34.0.

Table 2. Rhipicephalus sanguineus sensu stricto collected on dogs in Río Negro province, Patagonia, Argentina with results of the molecular detection of tick-borne rickettsial bacteria

L., larva; N., nymph; F., female; M., male.

^a positive sample in the gltA PCR assay

^b positive sample in the gltA and ompA PCR assays

Thirty-three tick pools were tested by the different PCR assays of which 3 were positive in the 16S rDNA PCR (Parola et al., 2000) for Anaplasmataceae bacteria (1 pool of larvae and 2 pools of nymphs), 2 in the gltA PCR (2 pools of females) and 1 in the ompA PCR (1 pool of females). From the three Anaplasmataceae positive samples, 2 partial 16S rDNA sequences belonging to A. platys could be generated: (I) a 273 bp fragment (GenBank acc. no. PP838794) from a pool of 10 nymphs of R. sanguineus s.s. and (II) a 301 bp fragment (GenBank acc. no. PP838795) from a pool of 8 R. sanguineus s.s. nymphs. Unfortunately, no sequence could be generated from the larval pool. The both obtained sequences were 100% identical to each other (273 bp/273 bp) and with partial sequences of A. platys strains detected in Argentina (GenBank acc. no. JX261979, KC525894, KF826282, KF826284) and other parts of the world (e.g. GenBank acc. no. KP717550, KP903296, KT359590, OP164858, OQ132527). Anaplasma platys is known as the causative agent of canine infectious cyclic thrombocytopenia (Diniz and Moura de Aguiar, 2022) and was firstly detected in R. sanguineus ticks from Northeastern Argentina in 2011 (Oscherov et al., 2011). Eiras et al. (2013) detected A. platys in dogs with suspected rickettsial disease in Buenos Aires province, Argentina. This rickettsial bacterium was also found in dogs from Buenos Aires City (Cicuttin et al., 2014), Salta (Dias Cordeiro et al., 2020), Córdoba and Santa Fe provinces (Mascarelli et al., 2016). Further, Cicuttin et al. (2015a, 2015b) showed the presence of A. platys in different R. sanguineus populations that belonged to the species R. linnaei in northern Argentina. In accordance with the results of the current and previous studies, it can be stated that both species – R. linnaei and R. sanguineus s.s. – could be potential vector of A. platys in Argentina. Laatamna et al. (2022) also detected A. platys in R. sanguineus s.s. ticks collected on dogs from Algeria, which is consistent with the results of the presented study. The vectorial competence of R. sanguineus s.s. for A. platys was demonstrated under laboratory conditions by Snellgrove et al. (2020).

Two partial sequences of the gltA gene from Rickettsia spp. were generated from female tick pools collected on different dogs. Both sequences (GenBank acc. no. PP856476, PP856477) showed sequence identities from over 99.24% to different sequences of R. massiliae, including some strains detected in Argentina. Further, an ompA partial sequence (GenBank acc. no. PP856478) from one of these gltA positive pools also could be generated and the BLAST comparison confirmed the identification of the sample as R. massiliae. This could be strengthened by the result of the phylogenetic analysis of the sequence (see Fig. 2).

Figure 2. Maximum-likelihood tree constructed from ompA partial sequences for different Rickettsia species. The partial generated in this study is written in bold letters. Numbers represents bootstrap support generated from 1.000. Genbank accession numbers are given in brackets. The alignment length was 432 bp and the General Time Reversible (GTR) substitution model with Gamma distribution and invariant sites was used.

Within the Maximum-likelihood tree constructed from ompA partial sequences for different Rickettsia species, the sequence generated in this study forms a clade with other strains of R. massiliae detected in Argentina and the type strain, which clearly separates (bootstrap value 96) from Rickettsia rhipicephali as the next related species. Rickettsia massiliae was previously detected in Argentina in canine blood samples and R. sanguineus ticks from Buenos Aires City (Cicuttin et al., 2004), Buenos Aires province (Cicuttin et al., 2014) and Mendoza province (Monje et al., 2016). This spotted fever group Rickettsia was described for the first time in 1993 (Beati and Raoult, 1993), and it is associated with ticks of the genus Rhipicephalus. Further, R. massiliae is related to infections in humans, although reports of human cases are rare (Portillo et al., 2015). So far, there is one confirmed case of disease in humans caused by R. massiliae (García-García et al., 2010) reported for Argentina. This association has public health relevance because R. sanguineus ticks are known to infest humans (Guglielmone et al., 2021).

Within this study we present the molecular detection of 2 rickettsial bacteria associated to R. sanguineus s.s. in a region of Argentina where no data about this topic were available so far. To our best knowledge the findings of A. platys and R. massiliae in R. sanguineus represent to most austral reports of these pathogens.