Rickettsial Infection in Ticks (Acari: Ixodidae) Collected on ... - BioOne

VECTOR/PATHOGEN/HOST INTERACTION, TRANSMISSION

Rickettsial Infection in Ticks (Acari: Ixodidae) Collected on Birds in Southern Brazil RICHARD C. PACHECO,1 MA´RCIA ARZUA,2 FERNANDA A. NIERI-BASTOS,3 JONAS MORAES-FILHO,3 ARLEI MARCILI,3 LEONARDO J. RICHTZENHAIN,3 DARCI M. BARROS-BATTESTI,4 AND MARCELO B. LABRUNA3

J. Med. Entomol. 49(3): 710Ð716 (2012); DOI: http://dx.doi.org/10.1603/ME11217

ABSTRACT The aim of the study was to evaluate rickettsial infection in ticks from wild birds of the Semidecidual and Atlantic Rainforest remnants of three municipalities of the State of Parana´, southern Brazil. Overall, 53 larvae and nymphs collected from birds were checked for the presence of Rickettsia DNA by molecular tests. Five tick species were tested: Amblyomma aureolatum (Pallas), Amblyomma calcaratum Neumann, Amblyomma longirostre (Koch), Amblyomma ovale Koch, and Amblyomma parkeri Fonseca and Aragaõ. A. longirostre ticks were infected with the spotted fever group agents Rickettsia amblyommii strain AL (32.3% infection rate) and Rickettsia parkeri strain NOD (5.9% infection rate). A new rickettsial genotype was detected in the tick A. parkeri (50% infection rate), which had never been reported to be infected by rickettsiae. Through phylogenetic analysis, this new genotype, here designated as strain ApPR, grouped in a cluster composed by different strains of Rickettsia africae, Rickettsia sibirica, and R. parkeri. We consider strain ApPR to be a new genotype of R. parkeri. This study reports for the Þrst time rickettsial infection in ticks from birds in southern Brazil. The role of migrating birds in the dispersal of these rickettsial strains should be considered in ecological studies of spotted fever group agents in Brazil. KEY WORDS Amblyomma longirostre, Amblyomma parkeri, Rickettsia amblyommii, Rickettsia parkeri, wild bird

Bacteria within the genus Rickettsia are obligate intracellular short rods, within the family Rickettsiaceae, order Rickettsiales, ␣-Proteobacteria. The Rickettsia genus is currently made of ⬇25 recognized species, and also contains several dozens of as-yet uncharacterized strains or tick amplicons. Most of these bacteria are associated with ticks, which are their vectors and reservoirs, but some are transmitted by lice, ßeas, or mites. Rickettsia species cause rickettsioses, which are among the oldest known arthropod-borne diseases (Fournier and Raoult 2007). 1 Corresponding author: Faculty of Agronomy and Veterinary Medicine, Federal University of Mato Grosso, Cuiaba´, MT, Brazil, Departamento de Cieˆ ncias Ba´sicas e Produç aõ Animal, Faculdade de Agronomia, Medicina Veterina´ria e Zootecnia, Universidade Federal de Mato Grosso. Av. Fernando Correˆ a da Costa, 2367. Boa Esperanç a. 78060-900. Cuiaba´, MT, Brazil (e-mail: [email protected]). 2 Laboratory of Parasitology, Capa õ da Imbuia Natural History Museum, Curitiba, PR, Brazil, Laborato´ rio de Parasitologia, Museu de Histo´ ria Natural Capaõ da Imbuia. R. Prof. Benedito Conceiç aõ, 407. Capaõ da Imbuia. 82810-080. Curitiba, PR, Brazil. 3 Faculty of Veterinary Medicine, University of Sa õ Paulo, Saõ Paulo, SP, Brazil, Departamento de Medicina Veterina´ria Preventiva e Sau´ de Animal, Faculdade de Medicina Veterina´ria e Zootecnia, Universidade de Saõ Paulo. Av. Prof. Dr. Orlando Marques de Paiva, 87. 05508-270. Saõ Paulo, SP, Brazil. 4 Laboratory of Parasitology, Institute Butantan, Sa õ Paulo, SP, Brazil, Laborato´ rio de Parasitologia, Instituto Butantan. Av. Vital Brasil, 1500. 05503-900. Saõ Paulo, SP, Brazil.

Traditionally, pathogenic rickettsiae were classiÞed into two groups: the typhus group (TG), containing R. prowazekii and R. typhi, which are transmitted by lice and ßeas, respectively, and the spotted-fever group (SFG), containing ⬎20 species, most of which have tick vectors (Raoult and Roux 1997). Other rickettsiae, such as R. bellii, the most common Rickettsia species reported in ticks from the New World (Labruna et al. 2007a), have shown antigenic and genetic particularities that preclude their inclusion in either the TG or SFG. Dissemination of SFG Rickettsia-infected ticks by migratory birds has been studied in Europe (SantosSilva et al. 2006, Elfving et al. 2010, Movila et al. 2011). In Brazil, the literature concerning rickettsial infection of ticks found on birds is restricted to recent studies in southeastern (Ogrzewalska et al. 2008, Ogrzewalska et al. 2009a), northern (Ogrzewalska et al. 2010), and northeastern (Ogrzewalska et al. 2011) regions of the country. Questions regarding the status of birds as reservoirs of rickettsiae have not been fully answered, in terms of the role of birds as sources of infection for naõ¨ve ticks. Therefore, it is important to know which rickettsiae are associated with bird ticks in nature (Elfving et al. 2010). Herein we evaluated rickettsial infection in ticks collected on wild birds of

0022-2585/12/0710Ð0716$04.00/0 䉷 2012 Entomological Society of America

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PACHECO ET AL.: RICKETTSIAL INFECTION IN TICKS FROM BRAZIL

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Fig. 1. Geographic location of the three municipalities of the current study in the state of Parana´, southern Brazil.

the semidecidual and Atlantic Rainforest remnants in Parana´ state, in the southern region of Brazil. Materials and Methods From March 2004 to February 2006, tick samples were obtained as part of a larger study on ecology of ticks infesting birds in southern Brazil (Arzua et al. 2010). Arzua et al. studied the diversity of ticks on wild birds of the Semidecidual and Atlantic Rainforest remnants of the State of Parana´, speciÞcally in the municipalities of Londrina (23⬚ 27⬘ S, 51⬚ 15⬘ WÐarea 1), Saõ Jorge do Oeste (25⬚ 41⬘ S, 53⬚ 03⬘ WÐarea 2), and Adriano´ polis (24⬚ 39⬘ S, 49⬚ 00⬘ WÐarea 3) (Fig. 1). Bird capture and sample collection in this work was approved by the Brazilian Institute of Natural Resources (IBAMA). For the current study, a total of 53 specimens, either nonengorged or partially engorged larvae and nymphs collected from a total of 21 bird species representing 13 families (Table 1), was preserved in absolute isopropanol, and then taken to the laboratory for identiÞcation of tick species and testing for the presence of Rickettsia DNA by molecular tests, as described below. For identiÞcation of tick species, we used molecular tools as described elsewhere (Ogrzewalska et al. 2009b). Brießy, immature specimen was processed for DNA extraction and individual extracts were used as template for polymerase chain reaction (PCR) assays using primers that amplify a ⬇460 bp of the tick mitochondrial 16S rDNA gene (Mangold et al. 1998). AmpliÞed products were puriÞed and DNA se-

quenced as previously described (Labruna et al. 2004b), and compared with NCBI Nucleotide BLAST searches (Altschul et al. 1990). Ticks were further tested individually by a battery of PCR using the following primer pairs, targeting fragments of three rickettsial genes: 1) primers CS-78 and CS-323, and CS-239 and CS-1069, which amplify two overlapping fragments (401 and 834 bp, respectively) of the citrate synthase gene (gltA) of all known Rickettsia species (Labruna et al. 2004b); 2) primers Rr190.70p and Rr190.602n, which amplify a 530 bp fragment of the 190 kDa outer membrane protein gene (ompA) of Rickettsia species belonging to the SFG (Regnery et al. 1991); 3) primers 120-M59 and 120 Ð 807, which amplify an 865 bp fragment of the 135 kDa outer membrane protein gene (ompB) (Roux and Raoult 2000). PCR products were DNA sequenced and submitted to BLAST analysis to determine similarities to other Rickettsia species (Altschul et al. 1990). In addition, sequences of a new genotype generated in this study were aligned by ClustalX (Thompson et al. 1997) with corresponding sequences downloaded from GenBank (Table 2), and manually adjusted with GeneDoc v. 2.6.01 (Nicholas et al. 1997). The network genealogy was inferred using Split Networks with Neighbor-Net method in Splits tree 4.10 (Huson and Bryant 2006). Internode support was estimated by performing 1,000 bootstrap pseudo replicates. Results The 53 ticks collected from birds were identiÞed to species level by molecular analysis as follows: Ambly-

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Table 1. Amblyomma ticks collected on birds, from Mar. 2004 to Feb. 2006, in three municipalities of the State of Paraná, Brazil, and Number (No.) of ticks infected by Rickettsia, as determined by PCR Birds Family Cardinalidae Conopophagidae Cuculidae Dendrocolaptidae Fringillidae Furnariidae Parulidae Pipridae Thamnophilidae Thraupidae Turdidae Tyrannidae Vireonidae Total

No. ticks tested by PCR (no. ticks infected by Rickettsia)

Species

Study area

A. ovale

Passerina brissonii Saltator similis Conopophaga lineata Crotophaga ani Xiphocolaptes albicollis Euphonia violacea Automolus leucophthalmus Basileuterus culicivorus Geothlypis aequinoctialis Chiroxiphia caudata Dysithamnus mentalis Pyriglena leucoptera Thamnophilus caerulescens Tachyphonus coronatus Trichothraupis melanops Turdus albicollis Turdus leucomelas Elaenia sp. Leptopogon amaurocephalus Pitangus sulphuratus Cyclarhis gujanensis

2 3 3 3 3 3 1 2 1,2,3 3 1 3 1 2,3 1 1 1,2 2 2 2 2

2 (0)

A. calcaratum

A. aureolatum

A. parkeri

2 (2) 1 (0) 1 (0) 1 (0) 1 (1) 1 (0) 2 (1) 3 (3) 4 (3) 1 (0) 1 (1) 2 (0)

1 (0) 4 (2) 1 (0) 2 (0) 3 (0) 2 (0)

1 (0)

3 (0)

omma ovale Koch, three nymphs (generated 16S rRNA sequences were 99.5% similar to GenBank accession AF541255); Amblyomma longirostre (Koch), 34 nymphs (100% similar to FJ424401); Amblyomma calcaratum Neumann, 11 nymphs (99.7% similar to FJ424400); Amblyomma aureolatum (Pallas), one nymph (97.6% similar to AF541254); and Amblyomma parkeri Fonseca and Aragaõ, four larvae (100% similar

A. longirostre

8 (2) 1 (0) 5 (0) 1 (0) 1 (0) 34 (13)

1 (0)

11 (0)

1 (0)

4 (2)

to EU805568). From 34 nymphs of A. longirostre tested by PCR, 13 (38.2%) collected on seven different species of birds yielded positive results by gltA, ompA, and ompB PCR assays, as well as two (50%) out of four larvae of A. parkeri collected on Chiroxiphia caudata (Table 1). The ompA (488 pb) and ompB (771 pb) products from 11 A. longirostre positive ticks from area two (three positive ticks) and area three (eight pos-

Table 2. GenBank accession numbers for the 190 kDa outer membrane protein (ompA) and 135 kDa outer membrane protein (ompB) partial sequences included in the phylogentic analyses (Fig. 2) of the present study Rickettsia species R. africae R. africae R. africae R. parkeri R. parkeri R. parkeri R. parkeri R. sibirica R. sibirica R. sibirica R. raoultii R. slovaca R. conorii R. conorii R. conorii R. rickettsii R. honei R. japonica R. aeschlimannii R. massiliae R. amblyommii R. amblyommii R. rhipicephali R. andeanae R. heilongjiangensis R. montanensis R. felis R. australis R. akari

Strain S AvR1/Kenya COOPERI NOD At24(⫽Maculatum) Atlantic rainforest BJ-90 Subsp. mongolotimonae Elanda-23/95 Subsp. israelensis Subsp. caspia Malish 7 Sheila Smith Subsp. marmionii Inha1 MTU5 AL TX116 HJ5 Argentina Extremiorientalis

ompA accession no.

ompB accession no.

EU622980 RSU43805 AF548338 AY362706 EU567180 EF102238 GQ855237 RSU43807 AF179365 DQ097082 EU036986 U43808 AY197565 RCU43794 AE006914 CP000848 DQ309096 DQ019319 Ñ CP000683 Ñ EF689733 U43803 Ñ AF179362 Ñ Ñ Ñ Ñ

AF123706 AF123720 Ñ Ñ EU567179 EF102239 GQ855236 AF123722 AY331393 DQ423364 EU036984 AF123723 AF123712 AY643093 Ñ CP000848 AF123711 AF123713 AF123705 AF123714 EU274657 AY375164 DQ865209 EF451003 AY280712 AF123716 AF182279 AF123709 AF123707

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itive ticks) were sequenced and were shown to be identical to each other, and 99.8% (487/488 for ompA) and 100% (756/756 for ompB) identical to the corresponding sequences of R. amblyommii strain AL (GenBank accession EU274656 and EU274657, respectively). Strain AL was originally isolated from A. longirostre ticks collected on birds in an Atlantic rainforest area in the State of Saõ Paulo, southeastern Brazil (Ogrzewalska et al. 2008). DNA sequences obtained from the ompA (491 bp) and ompB (784 bp) primer pairs of the two remaining PCR-positive A. longirostre nymphs, from area 2, were shown to be identical to each other, and 100% (491/491) and 99.8% (776/777), identical to the corresponding sequences of R. parkeri strain NOD in GenBank (EU567180 and EU567179, respectively). Strain NOD was previously isolated from Amblyomma nodosum Neumann ticks collected on birds in a fragmented region of Atlantic rainforest in the State of Saõ Paulo (Ogrzewalska et al. 2009a). No A. ovale, A. calcaratum, or A. aureolatum yielded positive results by PCR. Fragments of DNA of the three rickettsial genes (gltA, ompA, ompB) were successfully ampliÞed from two A. parkeri ticks from area 3. For each gene, DNA sequences obtained from the three ticks were identical to each other. Fragments of 1,094, 715, and 491 nucleotides of the gltA, ompB, and ompA genes, respectively, were obtained. The gltA partial sequence was 100% (1,093/1,093) identical to the corresponding sequence of R. sibirica (GenBank accession RSU59734), and 99.9% (1,092/1,093) identical to R. parkeri strain At24 (EF102236), isolated from Amblyomma triste Koch ticks in Brazil (Silveira et al. 2007). The ompB partial sequence was 99.7% (712/714) identical to the corresponding sequence of R. africae (CP001612) and 99.1% (708/714) identical to both R. sibirica (AF123722) and R. parkeri strain Atlantic rainforest (GQ855236), recently reported as the etiological agent of clinical cases of SFG rickettsiosis in Brazil (Spolidorio et al. 2010, Silva et al. 2011). The ompA partial sequences were 99.4% (488/491) identical to R. africae (CP001612), and 98.9% (486/491) to R. parkeri strain COOPERI (AY362706) isolated from Amblyomma dubitatum Neumann ticks in Brazil (Labruna et al. 2004b). Through phylogenetic analyses inferred from the partial sequences of ompA and ompB (Fig. 2), the rickettsial sequences generated from A. parkeri ticks, here designated as Rickettsia sp. ApPR, grouped in a cluster composed by different strains of R. africae, R. parkeri, and R. sibirica. This cluster was supported by high bootstrap value for ompB tree (84%), but relatively low for the ompA tree (70%). Partial sequences generated in this study were deposited in GenBank under the following accession numbers: JN126316 and JN126317 for ompA and ompB, respectively, of R. amblyommii from A. longirostre; JN126318 and JN126319 for ompA and ompB, respectively, of R. parkeri from A. longirostre; JN126320, JN126321, and JN126322 for gltA, ompA, and ompB, respectively, of R. parkeri strain ApPR from A. parkeri; JN573300 to JN573304 for mi-

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tochondrial 16S rRNA sequences of A. parkeri, A. aureolatum, A. calcaratum, A. longirostre, and A. ovale. Discussion This is the Þrst study to report rickettsial infection in ticks from birds in southern Brazil. Overall, 32.3 and 5.9% of the A. longirostre ticks were found to be infected by the SFG agents R. amblyommii (strain AL) and R. parkeri (strain NOD), respectively. Different strains of R. amblyommii have been reported infecting Amblyomma species in North, Central, and South America (Labruna et al. 2004a,b; Apperson et al. 2008; Ogrzewalska et al. 2008, 2010, 2011; Jiang et al. 2010; Bermu´ dez et al. 2011). While R. amblyommii is not currently recognized as a human or animal pathogen, there has been serological evidence for human or animal exposure with this agent in the United States, Panama, and in the Brazilian western Amazon (Labruna et al. 2007b, Apperson et al. 2008, Bermu´ dez et al. 2011). It has been proposed that some of the rickettsiosis cases reported as Rocky Mountain spotted fever (presumably caused by R. rickettsii) in the United States may have been caused by R. amblyommii (Apperson et al. 2008). Before the current study, R. amblyommii strain AL was reported infecting A. longirostre ticks collected on birds in the southeastern and northeastern regions of Brazil (Ogrzewalska et al. 2008, 2011), whereas R. parkeri strain NOD was reported solely in a western area of the state of Saõ Paulo, southeastern Brazil, infecting A. nodosum ticks collected from birds (Ogrzewalska et al. 2009a). Our Þndings indicate that the distribution area of these rickettsial strains is much broader than currently known. Moreover, the distribution of R. parkeri strain NOD in two tick species that parasitize passerine birds in Brazil, namely A. longirostre (current study) and A. nodosum (Ogrzewalska et al. 2009a), is suggestive of horizontal transmission of this rickettsial strain via infected birds. A new rickettsial strain, namely ApPR, is reported infecting A. parkeri ticks. Through phylogenetic analyses, this new strain clustered within a well-supported branch composed by different strains of R. parkeri, R. africae, and R. sibirica. These three Rickettsia species are etiological agents of very similar spotted fever entities (usually characterized by mild fever, inoculation scar, lymphadenopathy, and no fatality) on different continents (Paddock et al. 2004, Parola et al. 2005, Spolidorio et al. 2010). Recent reports regarding deÞnition of Rickettsia species proposed that a new Rickettsia species should not exhibit ⱖ99.9, 99.2, and 98.8% similarity for the gltA, ompB, and ompA genes, respectively, with the most homologous validated species (Fournier and Raoult 2009). If we adopt this procedure to the rickettsial strains of R. parkeri, R. africae, and R. sibirica that compose their clade in Fig. 1, several strains (including ApPR in relation to R. sibirica and R. africae, and also R. parkeri strain Atlantic rainforest in relation to R. africae, R. parkeri, and R. sibirica) exhibit similarity values greater than the cut-off values proposed above, indicating that they

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Fig. 2. Neighbor-Net based on the 190 kDa outer membrane protein gene (A); and 135 kDa outer membrane protein gene (B) partial sequences, showing the phylogenetic placement Rickettsia sp. strain ApPR among known Rickettsia species. The scale bar represents one substitutional change/100 nucleotide positions.

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belong as a whole to a single species. In fact, it has been proposed that R. parkeri and R. africae should be considered strains of a single species (Walker and Ismail 2008). Because R. parkeri, R. africae, and R. sibirica, albeit distributed in different continents and associated with different tick species, cause rickettsioses with similar symptoms in humans, they might be considered a single species represented by different strains. For now, we suggest strain ApPR to be a new strain of R. parkeri in Brazil, until further studies deÞne more precisely its taxonomic position. In the state of Parana´, southern region of Brazil, clinical cases of Brazilian spotted fever were recently conÞrmed by seroconversion using R. rickettsii antigens (Fiol et al. 2010, Freitas et al. 2010). None of these cases was fatal, contrasting to southeastern Brazil, where ⬇30% fatality rates have been observed among clinical cases in which R. rickettsii has been isolated (Angerami et al. 2006). Because cross-reaction antibodies between Rickettsia species are often observed, especially between SFG species, a serologic assay using a single rickettsial antigen is not sufÞcient to determine the Rickettsia species that elicited the serologic response (LaScola and Raoult 1997). For this reason, the SFG Rickettsia species involved in the BSF cases reported in southern Brazil, where mild febrile illness was reported, remains unknown (Labruna 2009). Herein we provide the Þrst reports of rickettsial infection in ticks from the state of Parana´, consisting of two strains of R. parkeri (strains NOD and ApPR), and R. amblyommii strain AL. The role of these strains as causative agents of the mild febrile cases of spotted fever in Parana´ remains to be investigated. Finally, the role of migrating birds in the dispersal of these rickettsial strains should also be considered in ecological studies of SFG agents in the New World. In fact, immature stages of A. longirostre have been reported in North America, where the ticks were transported from the Neotropical region via migrating birds (Scott et al. 2001). Acknowledgments We thank Sheila Oliveira de Souza for technical assistance in DNA sequencing. This work was supported by Conselho Nacional de Desenvolvimento Cientõ´Þco e Tecnolo´ gico (CNPq 478950/2004-7) and Fundaç aõ de Amparo a Pesquisa do Estado de Saõ Paulo (FAPESP).

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