Ticks (Acari: Ixodidae) Infesting Wild Birds in an Atlantic Forest Area in ...

VECTOR-BORNE DISEASES, SURVEILLANCE, PREVENTION

Ticks (Acari: Ixodidae) Infesting Wild Birds in an Atlantic Forest Area in the State of Sa˜ o Paulo, Brazil, with Isolation of Rickettsia from the Tick Amblyomma longirostre MARIA OGRZEWALSKA,1 RICHARD C. PACHECO,1 ALEXANDRE UEZU,2 FERNANDO FERREIRA,1 AND MARCELO B. LABRUNA1,3

J. Med. Entomol. 45(4): 770Ð774 (2008)

ABSTRACT During Þeld work in Nazare´ Paulista, state of Saõ Paulo, Brazil, we found 13 (56.5%) of 23 birds (mostly Passeriformes) to be infested by 28 larvae and 1 nymph of Amblyomma spp. Two larvae were reared to the adult stage, being taxonomically identiÞed as Amblyomma parkeri Fonseca and Aragaõ, whereas Þve larvae and one nymph were identiÞed as Amblyomma longirostre Koch. All six A. longirostre specimens were shown to be infected by rickettsia, as demonstrated by polymerase chain reaction (PCR) targeting two rickettsial genes (gltA and ompA) or isolation of rickettsia in cell culture from one of the ticks. This isolate was designated as strain AL, which was established in Vero cell culture and was molecularly characterized by DNA sequencing fragments of the rickettsial genes gltA, htrA, ompA, and ompB. Phylogenetic analyses inferred from ompA and ompB partial sequences showed a high degree of similarity of strain AL with Rickettsia sp. strain ARANHA, previously detected by PCR in A. longirostre ticks from Rondoˆ nia, northern Brazil. We conclude that strain AL is a new rickettsia genotype belonging to the same species of strain ARANHA, which are closely related to Candidatus ÕR. amblyommiiÕ. Further studies should elucidate if strains AL and ARANHA are different strains of Candidatus ÕR. amblyommiiÕ or are a new species. KEY WORDS Amblyomma longirostre, Amblyomma parkeri, Rickettsia amblyommii, birds, Atlantic forest

Bacteria of the genus Rickettsia are obligate intracellular organisms distributed worldwidely, with some species maintained in nature and transmitted to vertebrate hosts by arthropod vectors. The vast majority of the tick-borne Rickettsia species have been genetically and antigenically classiÞed into the spotted fever group (SFG), including ⬇20 valid species, some of them causing different types of spotted fever in different parts of the world (Roux and Raoult 2000). In South America, several species of ticks of the genera Argas, Ornithodoros, Ixodes, Amblyomma, and Hemaphysalis are found parasitizing birds (BarrosBattesti et al. 2006). However, the literature concerning rickettsial infection of ticks found on birds is scarce, and the role of these vertebrates as reservoirs or ampliÞer hosts for Rickettsia is still not clear. The tick Amblyomma longirostre Koch is widely distributed in southern Mexico, Central, and South America, in1 Faculty of Veterinary Medicine, University of Sa õ Paulo, Saõ Paulo, SP, Brazil. 2 Instituto de Pesquisas Ecolo ˆ ´ gicas (IPE), Nazare´ Paulista, SP Brazil. 3 Corresponding author: Departamento de Medicina Veterina ´ria Preventiva e Sau´ de Animal, Faculdade de Medicina Veterina´ria e Zootecnia, Universidade de Saõ Paulo, Av. Prof. Orlando Marques de Paiva, 87, Cidade Universita´ria, Saõ Paulo, SP 05508-270, Brazil (email: labruna@usp.br).

cluding Brazil (Barros-Battesti et al. 2006). The main hosts for the adult stage of A. longirostre are several species of porcupines (Coendou spp. and Sphiggurus spp.), whereas immature stages primarily parasitize passerine birds (Labruna et al. 2007), which can transport this tick northward (Keirans and Durden 1998). The purpose of this work was to add more information to the limited data about species of ticks parasitizing wild birds in Brazil and to report the Þrst isolation in cell culture of a rickettsia strain from the tick A. longirostre. Materials and Methods On 18 July 2006, wild birds were caught by mist nets in an Atlantic Forest area in Nazare´ Paulista (23⬚11⬘58⬙ S, 46⬚21⬘15⬙ W), State of Saõ Paulo, Brazil. Caught birds were identiÞed to the species and examined for the presence of ticks, which were collected and brought alive to the laboratory. Engorged larvae or nymphs were placed in an incubator at 25⬚C and RH 90% to allow them to molt to nymphs or adults, respectively. In some cases, nymphs obtained from the engorged larvae were allowed to feed on naive rabbits according to Pinter et al. (2002), to rear them to the adult stage. Each adult specimen obtained from an engorged nymph was used for identiÞcation of the

0022-2585/08/0770Ð0774$04.00/0 䉷 2008 Entomological Society of America

July 2008 Table 1.

OGRZEWALSKA ET AL.: TICKS AND Rickettsia IN BRAZIL Ticks collected on birds in an Atlantic Forest area in Nazaré Paulista, State of Sao Paulo, Brazil Birds

Order

771

Family

Piciformes Apodiformes

Bucconidae Trochilidae

Passeriformes

Thamnophilidae

Ticks Species

No. infested/ no. captured

Malacoptila striata Phaetornis pretrei Thalurania glaucops Thamnophilus caerulescens Dysithamnus mentalis

1/1 0/1 0/2 1/1 1/1

Conopophagidae

Conopophaga lineata

3/5

Furnariidae Tyrannidae

Synallaxis ruficapilla Leptopogon amaurocephalus

0/1 2/3

Platyrinchus mystaceus

1/1

Chiroxiphia caudata Basileuterus leucobleforus Trichothraupis melanops

2/2 0/2 2/2

Sporophila caerulescens

0/1

Pipridae Emberizidae

species of the former corresponding subadult tick (larva or nymph) that was collected on a bird. Isolation of Rickettsia in cell culture using the shell vial technique (Labruna et al. 2004b) was attempted from one adult tick obtained in the laboratory, originated from an engorged nymph collected on a bird. DNA extracted from infected cells were processed by a battery of polymerase chain reactions (PCRs) using previously described primer pairs targeting fragments of the rickettsial genes for citrate synthase (gltA) (Labruna et al. 2004a), 17-kDa genus-speciÞc protein (htrA) (Labruna et al. 2004a), outer membrane protein A (ompA) (Regnery et al. 1991), and outer membrane protein B (ompB) (Roux and Raoult 2000). All PCR products were puriÞed and directly sequenced in an automatic sequencer (model ABI Prism 310; Applied Biosystens/Perkin Elmer, Genetic, CA) according to the manufacturerÕs protocol. Partial sequences obtained were submitted to BLAST analysis (Altschul et al. 1990) to determine similarities to other Rickettsia species. Phylogenetic analyses were conducted using the program MEGA version 4 (Tamura et al. 2007). Partial DNA sequences obtained from ampliÞed PCR products of the genes ompA and ompB were aligned with the corresponding sequences of other Rickettsia species available in GenBank using the CLUSTAL W algorithm. For each gene analyzed, a phylogram was constructed by the neighbor-joining method using the Kimura-2-parameter model. ConÞdence values for individual branches of the resulting tree were determined by bootstrap analysis with 1,000 replicates. Other ticks that were identiÞed as A. longirostre were individually tested for the presence of Rickettsia by PCR targeting the ricketsial genes gltA and ompA, as described above. PCR products were sequenced and submitted to BLAST analysis to determine similarities to other Rickettsia species. We followed the protocol, which agrees with the Ethical Principles in Animal Research adopted by

Species A. longirostre A. parkeri A. longirostre Amblyomma sp. A. longirostre Amblyomma sp. A. longirostre Amblyomma sp. A. longirostre Amblyomma sp. Amblyomma sp. A. parkeri Amblyomma sp.

Number Larvae

Nymphs

1 Ñ Ñ 1 1 3 Ñ 10 Ñ 2 2 1 2 3 Ñ 1 1 Ñ

Ñ Ñ Ñ Ñ Ñ Ñ 1 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ

the Brazilian College of Animal Experimentation (COBEA) and which was approved by the Faculdade de Medicina Veterina´ria e Zootecnia /USP-Ethical Committee for Animal Research. Permits and approvals are on Þle in the ofÞce of M.B.L. Results A total of 23 birds representing 13 species from 8 families and 3 orders were captured. Thirteen birds (56.5%) were infested by ticks, made up of 28 larvae and 1 nymph, all belonging to the genus Amblyomma (Table 1). Two larvae were reared to the adult stage and were identiÞed as Amblyomma parkeri Fonseca and Araga˜ o according to Fonseca and Araga˜ o (1952), and Barros-Battesti et al. (2006). Five other ticks collected as engorged larvae molted to the nymphal stage, which were identiÞed as A. longirostre according to peculiar characteristics inherent to the nymphal stage of this species: scutum elongate and hypostome pointed (Keirans and Durden 1998, Labruna et al. 2007). The single engorged nymph was identiÞed as A. longirostre according to these peculiar characteristics, which was conÞrmed after this nymph molted to the adult stage in the laboratory. The remaining 21 larvae were identiÞed as Amblyomma sp. because they died in the laboratory before reaching the nymphal or adult stage. Bird species infested by each of these ticks are shown in Table 1. For logistic reasons, only ticks identiÞed as A. longirostre were tested for rickettsial infection. The single A. longirostre adult tick was processed for isolation of rickettsiae in cell culture by the shell vial technique. Rickettsiae were successfully isolated and established in Vero cells, with more than four passages, each reaching ⬎90% of infected cells, as determined by Gimenez staining. DNA of infected cells from the fourth passage generated PCR products for the four rickettsial genes evaluated (gltA, htrA, ompA, and ompB). PCR products of the gltA gene were se-

772

JOURNAL OF MEDICAL ENTOMOLOGY R. africae [U43790]

82

R. parkeri [U43802]

68 94 99

Vol. 45, no. 4

R. rickettsii [U43804] R. slovaca [U43808] R. conorii [U43806] R. japonica [U43795]

R. raoultii [DQ365801] R. aeschlimannii [DQ379981] R. rhipicephali [U43803]

99 98

R. massiliae [DQ212707]

Rickettsia sp. strain AL

86

Rickettsia sp. strain ARANHA (Brazil) [AY3602213] R. amblyommii strain AaR/SoCarolina (USA) [AF453408] 60

87

R. amblyommii strain TX051 (USA) [EF689731] 86

R. amblyommii strain APGEA17 (USA) [AY388928] R. amblyommii strain WB-82 (USA) [AY062007] R. amblyommii strain An13 (Argentina) [DQ517292] Rickettsia sp. strain Argentina [EF451004] R. montanensis [U43801] R. australis [AF149108]

0.01

Fig. 1. Neighbor-joining phylodendogram based on partial ompA sequences showing the phylogenetic placement of strain AL among Rickettsia species. Bootstrap support values ⱖ60 for phylogenetic groupings are indicated at the nodes. Numbers in brackets are GenBank accession numbers.

quenced, and after aligned, formed a consensus sequence of 1,090 bp that showed 99.5% (1,085/1,090) similarity to the corresponding sequence of R. raoultii (DQ365804) and Rickettsia sp. DnS14 (AF120028); part of this consensus sequence was 100% (772/772) identical to the available shorter sequence of Rickettsia sp. strain ARANHA (AY360216). The PCR product of the htrA gene generated a sequence 99.1% (480/ 484) similar to Rickettsia sp. strain ARANHA (AY360215). The PCR product of the ompA gene generated a sequence 99.6% (486/488) similar to the corresponding sequence of Rickettsia sp. strain ARANHA (AY360213). The PCR product of the ompB gene generated a sequence 99.3% (751/756) similar to Rickettsia sp. strain ARANHA (AY360214). This rickettsial isolate obtained from the A. longirostre adult specimen was designated as strain AL. The Þve other A. longirostre nymphs, collected as engorged larvae on birds, also showed to contain rickettsial DNA by both the gltA and ompA PCR assays. Their ompA products were sequenced, being identical to each other and to the corresponding sequence of strain AL. Thus, all six A. longirostre specimens identiÞed in this study were shown to contain Rickettsia sp. strain AL. Phylogenetic analysis inferred by ompA partial sequences placed strain AL with several available sequences of the Candidatus ‘R. amblyommiiÕ, being closest to Rickettsia sp. strain ARANHA under 86% bootstrap support (Fig. 1). Phylogenetic analysis inferred by ompB partial sequences placed strain AL in the subgroup with Rickettsia sp. strain Argentina, and

CandidatusÔR. amblyommiiÕ; however, again strain AL was closest to Rickettsia sp. strain ARANHA, under 92% bootstrap support (Fig. 2). Voucher specimens of ticks collected in this study have been deposited in the tick collection “Coleç aõ Nacional de Carrapatos (CNC)” of the University of Saõ Paulo (accession no. 998). GenBank nucleotide sequence accession numbers for the partial DNA sequences of Rickettsia sp. strain AL generated in this study are EU274654, EU274655, EU274656, and EU274657 for the gltA, htrA, ompA, and ompB genes, respectively. Discussion This study reported immature stages of both A. longirostre and A. parkeri infesting birds in Nazare´ Paulista, state of Saõ Paulo. A. longirostre has been previously reported infesting various bird species (mostly Passeriformes species), including all the Þve species reported here (Labruna et al. 2007). A. parkeri is a rare tick species that until very recently was known only from the type specimens, described in 1952 (Fonseca and Aragaõ 1952, Barros-Battesti et al. 2006). This study found two A. parkeri larvae parasitizing two Passeriformes species for the Þrst time. These ticks were identiÞed with certainty because they were reared to the adult stage in the laboratory. We found that all ticks identiÞed as A. longirostre (Þve larvae, one nymph) were infected with a new strain of SFG Rickettsia, designed as strain AL. Because these ticks were collected fully engorged, it was not

July 2008

OGRZEWALSKA ET AL.: TICKS AND Rickettsia IN BRAZIL

99 79

773

R. africae [AF123706] R. parkeri [AF123717]

R. sibirica [AF123722] R. conorii [AY643093] 99 R. slovaca [AF123723] R. rickettsii [X16353 ] 90 62 R. honei [AF123711] R. japonica [AF123713] R. heilongjiangensis [AY260451] 100 100 83 R. hulinensis [AY260452] R. montanensis [AF123716] Rickettsia sp. strain Koala [EF212861] 73 R. massiliae [AF123714] 64 R. rhipicephali [AF123719 ] 67 90 R. raoultii [DQ365798] R. aeschlimannii [AF123705] 66 Rickettsia sp. strain Argentina [EF451003] R. amblyommii [AY375164] 98 Rickettsia sp. strain ARANHA [AY360214] 74 92 Rickettsia sp. strain AL R. felis [AF182279] R. akari [AF123707] 79 100 R. australis [AF123709] R. typhi [L04661] R. prowazekii [AF161079] 100 83

0.02

Fig. 2. Neighbor-joining phylodendogram based on partial ompB sequences showing the phylogenetic placement of strain AL among rickettsial species. Bootstrap support values ⱖ60 for phylogenetic groupings are indicated at the nodes. Numbers in brackets are GenBank accession numbers.

necessary to feed them in the laboratory, discarding any possibility of accidental rickettsial infection during rabbit infestations. Phylogenetic analyses inferred by fragments of either ompA or ompB genes grouped strain AL in a single cluster with strain ARANHA and available sequences of CandidatusÔR. amblyommiiÕ, under high bootstrap support (87 or 74%, respectively). In all analyses, the sequences of strain AL were closest to Rickettsia sp. strain ARANHA, which was previously recorded from adults of A. longiroste parasitizing porcupine (Coendou prehensilis) in the state of Rondoˆ nia, Western Brazilian Amazon (Labruna et al. 2004b). We did not perform phylogenetic analysis with gltA and htrA partial sequences because previously studies showed that these genes are not very informative for phylogeny of SFG rickettsia and that less conservative genes such as ompA and ompB are more indicated for comparisons of SFG species (Roux et al. 1997, Fournier et al. 2003). A recent report of gene sequenceÐ based criteria for identiÞcation of new Rickettsia species proposed that, to be classiÞed as a new Rickettsia species, an isolate should not exhibit more than one of the following degrees of nucleotide similarity with the most homologous validated species: ⱖ99.8 and ⱖ99.9% for the rrs and gltA genes, respectively, and, when ampliÞable, ⱖ98.8, ⱖ99.2, and ⱖ99.3% for the ompA, ompB, and sca4 genes, respectively (Fournier et al. 2003). Comparisons between partial sequences of strains AL and

ARANHA showed similarities above these cut-off values for gltA, ompA, and ompB partial sequences, indicating that these two strains belong to the same species. Comparing strain AL (and also strain ARANHA; data not shown) with other rickettsiae, the only similarity found above the cut-off value was with the ompA partial sequence of CandidatusÔR. amblyommiiÕ. Unfortunately, there are no rrs and sca4 sequences available for comparisons. Thus, at this moment, we conclude that strain AL is a new rickettsia genotype belonging to the same species of strain ARANHA, which is found infecting the tick A. longirostre. Further studies should elucidate if strains AL and ARANHA are different strains of CandidatusÔR. amblyommiiÕ or are a new species.

Acknowledgments We thank two anonymous reviewers for valuable suggestions to improve this manuscript. This work was supported by FAPESP (grant to M.B.L. and scholarship to M.O.) and CNPq (Academic Career Scholarship to M.B.L. and F.F.).

References Cited Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403Ð 410.

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Barros-Battesti, D. M., M. Arzua, and G. H. Bechara. 2006. Carrapatos de importaˆncia me´ dico-veterina´ria da Regiaõ Neotropical: Um guia ilustrado para identiÞcaç aõ de espe´ cies. Saõ Paulo. Vox/International Consortium on Ticks and Tick-borne Diseases (ICTTD-3), Butantan, Brazil. Fonseca, F., and H. B. Araga˜ o. 1952. Notas de ixodologõá. II. Uma nova espe´ cie do geˆ nero Amblyomma e uma nova espe´ cie do geˆ nero Ixodes (Acari: Ixodidae). Mem. Inst. Oswaldo. Cruz. 50: 713Ð726. Fournier, P. E., J. S. Dumler, G. Greub, J. Zhang, Y. Wu, and D. Raoult. 2003. Gene sequence-based criteria for identiÞcation of new Rickettsia isolates and description of Rickettsia heilongjiangensis sp. nov. J. Clin. Microbiol. 41: 5456 Ð5465. Keirans, J. E., and L. A. Durden. 1998. Illustrated key to nymphs of the tick genus Amblyomma (Acari: Ixodidae) found in the United States. J. Med. Entomol. 35: 489 Ð 495. Labruna, M. B., J. W. McBride, D. H. Bouyer, L.M.A. Camargo, E. P. Camargo, and D. H. Walker. 2004a. Molecular evidence for a spotted fever group Rickettsia species in the tick Amblyomma longirostre in Brazil. J. Med. Entomol. 41: 533Ð537. Labruna, M. B., T. Whitworth, M. C. Horta, D. H. Bouyer, J. W. McBride, A. Pinter, V. Popov, S. M. Gennari, and D. H. Walker. 2004b. Rickettsia species infecting Amblyomma cooperi ticks from an area in the state of Sao

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Paulo, Brazil, where Brazilian spotted fever is endemic. J. Clin. Microbiol. 42: 90 Ð98. Labruna, M. B., L. F. Sanfilippo, C. Demetrio, A. C. Menezes, A. Pinter, A. A. Guglielmone, and L. F. Silveira. 2007. Ticks collected form birds in the state of Saõ Paulo, Brazil. Exp. Appl. Acarol. 43: 147Ð160. Pinter, A., M. B. Labruna, and J.L.H. Faccini. 2002. The sex ratio of Amblyomma cajennense (Acari: Ixodidae) with notes on the male feeding period in the laboratory. Vet. Parasitol. 105: 79 Ð 88. Regnery, R. L., C. L. Spruill, and B. D. Plikaytis. 1991. Genotypic identiÞcation of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J. Bacteriol. 173: 1576 Ð1589. Roux, V., and D. Raoult. 2000. Phylogenetic analysis of members of the genus Rickettsia using the gene encoding the outer membrane protein rOmpB (ompB). Int. J. Syst. Evol. Microbiol. 50: 1449 Ð1455. Roux, V., E. Rydkina, M. Eremeeva, and D. Raoult. 1997. Citrate synthase gene comparison, a new tool for phylogenetic analysis and its application for the rickettsiae. Int. J. Syst. Bacteriol. 47: 252Ð261. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596 Ð1599. Received 17 December 2007; accepted 24 March 2008.