Molecular Detection of the Human Pathogenic Rickettsia sp. Strain Atlantic Rainforest in Amblyomma dubitatum Ticks from Argentina

Introduction

In South America, clinical cases of human rickettsioses caused by tick-borne rickettsiae were reported in Argentina, Brazil, Colombia, and Uruguay (for review, see Parola et al. 2013). In Argentina, six species of Rickettsia were found infecting ticks, namely, R. rickettsii, R. parkeri, R. massiliae, R. bellii, ‘Candidatus R. amblyommii’, and ‘Candidatus R. andeanae’ (Parola et al. 2013). Of these, R. rickettsii, R. parkeri, and R. massiliae are associated with human diseases (Parola et al. 2013). The respective vectors of these three pathogens in Argentina are the ticks Amblyomma cajennense sensu lato (s.l.), Amblyomma triste, and Rhipicephalus sanguineus s.l. (Paddock et al. 2008, Monje et al. 2014, Cicuttin et al. 2014). In the past few years, new strains of Rickettsia, some of them closely related to R. parkeri, were described in Brazil infecting Amblyomma nodosum (strain NOD), Amblyomma ovale (strain Atlantic rainforest), and Amblyomma dubitatum (strains COOPERI and Pampulha) ticks (Parola et al. 2013). Of these, Rickettsia sp. strain Atlantic rainforest is the only one currently associated with human cases of rickettsiosis (Spolidorio et al. 2010).

Amblyomma dubitatum feeds predominantly on capybaras (Hydrochoerus hydrochaeris), but all stages of this tick species were recorded biting humans (Labruna et al. 2007). In northeastern Argentina, capybaras are present at high densities living close to farms and urban settlements, resulting in an extensive human–wildlife interface that may represent a potential risk to public health. Due to the possible role of A. dubitatum as an enzootic vector of potentially pathogenic rickettsiae, this study evaluated the infection with Rickettsia species of A. dubitatum ticks collected from vegetation and feeding from capybaras in northeastern Argentina.

Material and Methods

Ticks were collected at five localities from northeastern Argentina (Table 1). Questing larvae, nymphs, and adults were collected from the vegetation using flannel cloth flags in four areas of Corrientes province. Feeding ticks were obtained from wild capybaras (around ten) belonging to populations close to Romang city, Santa Fe province (Table 1). Of these latter, only males, nonengorged females, and nonengorged nymphs were analyzed. Ticks were determined by S.N. and A.A.G. by using standard taxonomic keys and morphological descriptions. All adults and nymphs were analyzed individually, and larvae were divided in pools according to collection site. Ticks were processed for DNA extraction by a boiling technique (Monje et al. 2014) and tested for rickettsial infection by conventional PCR targeting gene gltA using primers CS-78/CS-323 (Labruna et al. 2004). Positive samples were further tested with primers targeting genes gltA (larger fragment) and ompA (Labruna et al. 2004). Reactions were performed in triplicate, and PCR products were purified and sequenced. Resultant sequences were compared to GenBank data by BLAST analysis.

Results

Most of the ticks were identified as A. dubitatum (119 adults [78 males, 41 females], 176 nymphs, and 79 larvae). One nymph and two larvae were identified as A. triste, and one nymph was identified as Haemaphysalis juxtakochi (all questing). Eight adult ticks, six nymphs, and eight larvae pools tested positive for gltA PCR using primers CS-78/CS-323 (Table 1). The few A. triste and H. juxtakochi samples were PCR negative. Only three adult ticks collected on H. hydrochaeris from Romang (Ad106 in May, 2012; Ad144 and Ad170 in November, 2012) were positive for both the larger fragment of gltA and ompA genes. Moreover, only gltA PCR products were obtained in the rest of the positive samples (n=19) that were 100% identical to R. bellii (acc. no. CP000087). Sequences from sample Ad106 were identified as Rickettsia sp. strain Atlantic rainforest, because its gltA (787 bp) and ompA (592 bp) gene fragments were 100% equal to corresponding sequences (JQ906783 and JQ906784, respectively). Sequences from Ad144 and Ad170 were 100% identical to each other for the rickettsial gen ompA and were identified as Rickettsia sp. strain COOPERI. BLAST analysis showed that the ompA sequence from ticks Ad144 and Ad170 (592 bp) was 99.8% identical to strain COOPERI (AY362706). The gltA sequence (758 bp) from tick Ad170 was 99.9% identical to strain COOPERI (AY362704) and 99.7% identical to R. parkeri (CP003341). Surprisingly, sample Ad144 was co-infected with R. bellii because its gltA fragment was 100% identical to R. bellii (CP000087). Novel sequences were deposited in GenBank (KM116015-KM116018).

Discussion

In the present study, we report the presence of the human pathogen Rickettsia sp. strain Atlantic rainforest (Spolidorio et al. 2010) for the first time in Argentina. Moreover, our findings posit A. dubitatum as a potential vector for strain Atlantic rainforest, which was previously reported infecting A. ovale ticks in southeastern Brazil (Szabo et al. 2013).

Of five A. dubitatum populations examined, R. bellii was found in four, with prevalences that ranged from 3.0% to 47.1%. Rickettsia sp. strain Atlantic rainforest and Rickettsia sp. strain COOPERI were found only in Romang, where the prevalences were 1.0% and 2.0%, respectively (Table 1). No evidence of rickettsial DNA was observed in the samples from site “Mburucuyá,” although over 100 ticks were analyzed. Previous studies failed to detect Rickettsia sp. strain Atlantic rainforest in various populations of A. dubitatum in Brazil, even when thousands of adult ticks were tested (Labruna et al. 2004, Pacheco et al. 2009, Almeida et al. 2011). In those studies, A. dubitatum ticks were infected chiefly by R. bellii, Rickettsia sp. strain COOPERI, and Rickettsia sp. strain Pampulha (Labruna et al. 2004, Pacheco et al. 2009, Almeida et al. 2011). In addition, R. parkeri was recently detected in A. dubitatum ticks from Uruguay (Lado et al. 2014). Altogether, these results suggest that different populations of A. dubitatum throughout Argentina, Brazil, and Uruguay are capable of maintaining a variety of rickettsiae, including the human pathogens Rickettsia sp. strain Atlantic rainforest and R. parkeri. However, because the evidence of the presence of pathogenic Rickettsia was obtained from A. dubitatum ticks feeding on hosts, these results should be complemented with data from free-living ticks to confirm the vector capacity of A. dubitatum ticks.

Finally, further studies are warranted to elucidate the determinants of the distinctive geographical distribution of Rickettsia spp. observed in the region and to determine the potential pathogenicity and clinical significance of Rickettsia sp. strain COOPERI. Moreover, the fact that Rickettsia sp. strain Atlantic rainforest is pathogenic (Spolidorio et al. 2010) and A. dubitatum can parasitize humans in all its feeding stages (Labruna et al. 2007) indicates that human cases of spotted fever caused by this pathogen may occur in northeastern Argentina.