CONICET | Buscador de Institutos y Recursos Humanos

Introduction: Cases of rickettsioses produced by the genus Rickettsia have been described in several countries of South America in the last 20 years. In Argentina, seven Rickettsia species have been reported: Rickettsia rickettsii, Rickettsia parkeri, Rickettsia massiliae, Rickettsia bellii, Candidatus Rickettsia amblyommii, and Candidatus Rickettsia andeanae, all associated with ticks, and Rickettsia felis associated with fleas. Of these, R. rickettsii and R. parkeri are the etiological agents of Rocky Mountain spotted fever and eschar-associated spotted fever, respectively; which are transmitted in Argentina by the ticks Amblyoma cajennense sensu lato and Amblyomma triste. 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. Of these new strains, only Rickettsia sp. strain Atlantic rainforest has been reported as a human pathogen. Amblyomma dubitatum feeds typically on capybaras (Hydrochoerus hydrochaeris), which are present at high densities in northeastern Argentina. In this region A. cajennense does not occur, and capybaras are found infested mainly by A. dubitatum. Herein we report for the first time in Argentina the presence of Rickettsia sp. strain Atlantic rainforest and describe A. dubitatum as a new vector of this pathogen. In addition, we report the presence of Rickettsia sp. strain COOPERI in northeastern Argentina. Material and Methods: Ticks were collected both questing from five areas of Corrientes province (28°01S 58°01W, 28°39S 57°26W, 29°09S 58°04W, 28°50S 57°34W, 28°36S 58°24W) and feeding from two populations of free-ranging capybaras; one in Esteros del Iberá, Corrientes province (28°39?S 57°26?W) and the other one in the surroundings of Paraná River, Santa Fe province (29°30S 59°44W). Questing ticks were collected from the vegetation using flannel cloth flags. Engorged ticks (nymphs) were left in an incubator for molting to adults. Non-engorged and questing ticks were stored in 70% ethanol and brought to the laboratory for identification. Larvae were divided in 22 pools of 2 5 ticks each according to collection site. All ticks were processed for DNA extraction by a boiling technique. Samples were screened for rickettsial infection by real-time PCR using primers CS-5/CS-6, targeting a 147-bp fragment of the gene gltA. Samples yielding a Ct ≤ 30 by this PCR were further tested with primers targeting genes gltA (larger fragment), and ompA. PCR products were purified and sequenced. Results: Most of the ticks were identified as A. dubitatum (156 adults {97 males, 59 females}, 268 nymphs and 91 larvae). One nymph and two larvae were identified as A. triste and one nymph was identified as Haemaphysalis juxtakochi (all questing). Using real time PCR, high levels of rickettsial DNA were detected in 12 adult ticks (from 3.5×105 to 1.7×107 gltA copies/tick), 9 nymphs (from 7.0×104 to 5.0×105 gltA copies/nymph) and 12 larvae pools (from 1.0×104 to 2.0×105 gltA copies/pool). The few A. triste and H. juxtakochi samples were PCR negative. Ticks that presented high levels of rickettsial DNA were screened for the presence of a larger fragment of gltA and ompA genes. PCR products of the expected sizes were obtained for both genes studied in three samples (Ad106, Ad144, Ad170). Moreover, only gltA PCR products were obtained for five samples which in all cases were 100% identical to the corresponding sequence of R. bellii (CP000087). Sequences generated from sample Ad106 were identified as Rickettsia sp. strain Atlantic rainforest, since its gltA (787 bp) and ompA (592 bp) gene fragments were 100% equal to corresponding sequences (GQ855235, GQ855237, respectively). Sequences generated from samples Ad144 and Ad170 were identified as Rickettsia sp. strain COOPERI. Sequences from these ticks were 100% identical to each other for the rickettsial gene ompA. BLAST analysis showed that the ompA sequence generated from ticks Ad144 and Ad170 (592 bp) was 99.8% (490/491) identical to Rickettsia sp. strain COOPERI (AY362706). The gltA sequence (758 bp) generated from tick Ad170 was 99.9% (757/758) identical to Rickettsia sp. strain COOPERI (AY362704). Surprisingly, sample Ad144 was shown to be co-infected with R. bellii since the gltA fragment obtained from this sample was 100% identical to the corresponding sequence of R. bellii (CP000087). Discussion: Herein we report the presence of the human pathogen Rickettsia sp. strain Atlantic rainforest for the first time in Argentina. Moreover, we describe A. dubitatum as a potential vector for Rickettsia sp. strain Atlantic rainforest which was previously reported infecting A. ovale ticks in southeastern Brazil. Our results showed rather low infection rates of Rickettsia sp. strain Atlantic rainforest (1.0%) and Rickettsia sp. strain COOPERI (4.0%), and variable infection rates of R. bellii (from 3.8% to 84.6%) among the A. dubitatum populations studied. Interestingly, previous studies failed to detect Rickettsia sp. strain Atlantic rainforest in different populations of A. dubitatum in Brazil, although thousands of adult ticks were tested. In those studies, A. dubitatum ticks were shown to be infected chiefly by R. bellii, Rickettsia sp. strain COOPERI and Rickettsia sp. strain Pampulha. Altogether, these results suggest that different populations of A. dubitatum throughout Argentina and Brazil are capable of maintaining a variety of rickettsiae, conferring to A. dubitatum ticks a potential role in the ecology of Rickettsia in South America. Finally, further studies are necessary to determine the potential pathogenicity and clinical significance of Rickettsia sp. strain COOPERI. Moreover, the facts that strain Atlantic rainforest is pathogenic and A. dubitatum can parasitize humans in all its feeding stages indicates that human cases of spotted fever may be occurring in northeastern Argentina.