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A Third Amblyomma Species and the

First Tick-Borne Rickettsia in Chile

Article in Journal of Medical Entomology · January 2012

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 SHORT COMMUNICATION

A Third Amblyomma Species and the First Tick-Borne Rickettsia in Chile

KATIA ABARCA,1 JAVIER LÓPEZ,2 GERARDO ACOSTA-JAMETT,3 PAULINA LEPE,2
JOÃO FÁBIO SOARES,4 AND MARCELO B. LABRUNA4,5

J. Med. Entomol. 49(1): 219Ð222 (2012); DOI: http://dx.doi.org/10.1603/ME11147

ABSTRACT During November 2010, three ticks were collected from three dogs living in the rural
area of Arica, northern Chile. Morphological analyses of the ticks in the laboratory revealed that they
were most similar to Amblyomma maculatum Koch and Amblyomma triste Koch. However, because
of unique metatarsal spurs, neither of the Chilean specimens could be assigned with certainty to A.
maculatum or A. triste, based on external morphology. The mitochondrial 16S rRNA gene partial
sequences obtained from two Chilean specimens were 99.5% identical to A. triste from Uruguay, and
99.0% identical to A. maculatum from the United States. Through phylogenetic analysis inferred from
partial 16S rRNA sequences, the Chilean specimens were classiÞed as A. triste. Molecular analyses also
showed that one of the three Chilean ticks was infected by Candidatus ÔRickettsia andeanaeÕ. These
Þndings extend the geographical distribution of A. triste to Chile, where no tick-associated rickettsia
had been reported previously.

KEY WORDS Amblyomma triste, Amblyomma maculatum, Rickettsia andeanae, Chile

The South American tick fauna is represented by ⬇145
species, divided into two families, namely Argasidae
(52 species) and Ixodidae (93 species). As much as 51
(55%) of the Ixodidae species reported in South Amer-
ica belong to the genus Amblyomma (Guglielmone et
al. 2003, 2010). These include species of major medical
importance, with human biting behavior and trans-
mission of tick-borne diseases, especially spotted fever
rickettsioses (Labruna 2009).
Because of its particular geographic conditions,
Chile is the only South American country where the
genus Amblyomma has been reported to be poorly
represented, with only two established species,
namely Amblyomma tigrinum Koch, and Amblyomma
parvitarsum Neumann (González-Acuña and Gugliel-
mone 2005). Although there has been an increasing
number of tick-borne rickettsiae identiÞed during the
last decades among South American countries
(Labruna 2009, Romer et al. 2011), rickettsia infecting
ticks in Chile never have been reported.
Herein, we report the presence of a third Ambly-
omma species in Chile. One of the ticks also was found
to be infected by a recently described spotted fever
group Rickettsia, which is to our knowledge, the Þrst
record of a rickettsial infection in a Chilean tick.
1 Facultad de Medicina, PontiÞcia Universidad Católica de Chile,
Santiago, Chile.
2 Hospital Veterinario Puente Alto, Santiago, Chile.
3 Instituto de Medicina Preventiva Veterinaria y Programa de In-
vestigación Aplicada en Fauna Silvestre, Facultad de Ciencias Vet-
erinarias, Universidad Austral de Chile, Valdivia, Chile.
4 Faculdade de Medicina Veterinária e Zootecnia, Universidade de
Materials and Methods
During November 2010, one of us (J. L.) collected
three ticks from three dogs (each tick from one dog)
living in the rural area of Arica (18⬚ 28⬘ S, 70⬚ 18⬘ W),
northern Chile, only 10 km from the border with
Peru. These three dogs were reared unrestrained,
with free outdoor access, and had no recent history
of leaving the rural area of Arica. The ticks were
brought to the laboratory, where taxonomic iden-
tiÞcation of the ticks to species level was attempted
based on current literature (Estrada-Peña et al.
2005), and by comparisons with adult specimens of
Amblyomma maculatum Koch from Colombia, and
Texas, and Amblyomma triste Koch from the state of
Mato Grosso do Sul, Brazil, both available in the
tick collection “Coleção Nacional de Carrapatos”
(CNC) of the University of São Paulo. Two ticks
(one male, one female) had their internal contents
removed through a slight perforation of the poste-
rior margin of the idiosoma, by using a 21-gauge
sterile needle. The internal contents of each of the
two ticks was processed separately for DNA extrac-
tion by using the DNeasy Tissue Kit (Qiagen, Chats-
worth, CA), and then processed by polymerase
chain reaction (PCR) protocols targeting portions
of the tick mitochondrial 16S rRNA gene (Mangold
et al. 1998), the rickettsial gltA gene (Labruna et al.
2004), and the rickettsial ompA gene (Roux et al.
1996). PCR products were sequenced in an automatic
sequencer (Applied Biosystems/PerkinElmer, model

São Paulo, São Paulo, SP, Brazil. ABI Prism 310 Genetic, Foster City, CA) according to
5 Corresponding author, e-mail: labruna@usp.br. the manufacturerÕs protocol. Partial sequences ob-

0022-2585/12/0219Ð0222$04.00/0 䉷 2012 Entomological Society of America

220 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 49, no. 1

Fig. 1. A. triste specimens from northern Chile. (A) Female dorsal view. (B) Male dorsal view. (C) Female coxae IÐIV.
(D) Chitinous tubercles on female festoons. (E) Female metatarsus II. (F) Female metatarsus III. (G) Male metatarsus II.
(H) Male metatarsus III. (I) Male metatarsus IV. Each of metatarsus IIÐIV has a strong spine (black arrow) and a weaker
spine, resembling a strong seta (white arrow).

tained were submitted to BLAST analysis to deter-
mine the closest similarities.
For phylogenetic analyses, the partial 16S rRNA
consensus DNA sequence of the Chilean ticks, and the
corresponding sequences of A. maculatum, A. triste, A.
tigrinum, and A. parvitarsum available in GenBank
were aligned manually using GeneDoc software. A
phylogenetic tree was inferred by the maximum par-
simony (MP) method by using PAUP* 4.0b10 (Swof-
ford 2002) with 1,000 replicates of random addition
taxa and TBR branch swapping; all positions were
weighted equally. The sequence of A. parvitarsum was
used as the outgroup.
Results
Through morphological analyses, the three ticks
collected from dogs in Arica were shown to be most
similar to A. maculatum and A. triste, as shown by the
scutal ornamentation, coxal spur patterns, and pres-
ence of tubercles at the postero-internal angle of all
festoons, except for the middle one (Fig. 1). However,
because of unique metatarsal spurs, neither of the
Chilean specimens could be assigned with certainty to
A. maculatum or A. triste, based on external morphol-
ogy. Although the A. maculatum specimens from Co-
lombia and Texas typically were shown to have a pair
of stout ventral spurs on the distal extremity of meta-
tarsi II, III, and IV, and the A. triste specimens from
Brazil typically were shown to have one stout meta-
tarsal spur and a long seta instead of a second spur, the
specimens from Chile were shown to have an inter-
mediate spur pattern, characterized by a stout meta-
tarsal spur and a second spur that was not as stout as
the second one of A. maculatum, but at the same time,
much broader than the long metatarsal seta of A. triste
(Fig. 2).
The mitochondrial 16S rRNA gene partial se-
quences obtained from the two Chilean specimens
(one male, one female) were shown to be identical to
each other, and after Blast analysis, were shown to be
99.5% (397/399) identical to A. triste from Uruguay
(AY498563), and 99.0% (404/408) identical to A.
maculatum from the United States (L34318). Through
phylogenetic analysis, the partial 16S rRNA consensus
DNA sequence of the Chilean ticks were grouped with
A. triste under 98% bootstrap support, having A. macu-
latum as sister sequences (Fig. 3). Based on these
genetic results, the Chilean specimens were classiÞed
as A. triste.
Only the female Chilean tick yielded PCR ampli-
cons targeting the rickettsial genes gltA and ompA. The
gltA partial sequence (350-bp) was 100% identical to
Candidatus ÔRickettsia andeanaeÕ and also 100% to
Rickettsia sp strain Argentina (EF451004, EU826513).
The ompA partial sequence (587-bp) was 100% iden-
tical to a Rickettsia endosymbiont of A. maculatum
January 2012 ABARCA ET AL.: Amblyomma AND TICK-BORNE RICKETTSIA IN CHILE 221

Fig. 2. Spines on metatarsus IV of female ticks. (A) A. maculatum from Colombia. (B) A. triste from northern Chile. (C) A.
triste from Brazil. Strong spine (black arrow), weaker spine, resembling a strong seta (white arrow), and seta (white arrow head).

(EF372578), and also 100% to Rickettsia sp strain Ar-
gentina (EF451004, EU826513).
The Chilean A. triste specimens of the current study
have been deposited in the CNC tick collection, under
the accession number CNC-1918. GenBank nucleo-
tide sequence accession numbers for the partial se-
quences generated in this study are JN180848 for par-
tial sequences of A. triste 16S rRNA mitochondrial
gene, and JN180849, and JN180850 for partial se-
quences of Candidatus ÔR. andeanaeÕ gltA, and ompA
genes, respectively.
Discussion
The taxa A. maculatum, A. triste, and A. tigrinum
were described as valid species by Koch (1844), based
on adult specimens from the United States, Uruguay,
and Brazil, respectively. However, Neumann (1899)
considered these three taxa to belong to a single spe-
cies, namely A. maculatum, which was adopted by
subsequent authors until the study of Kohls (1956),
who revalidated the three species based on morpho-
logical characters. The Chilean specimens of the cur-
rent study were shown to have an intermediate spur
pattern, characterized by a stout metatarsal spur and
a second spur that was not as stout as the second one
from A. maculatum, but at the same time, much
broader than the long seta of A. triste. However, these
specimens were shown to be genetically closest to A.
triste, allowing us to classify them as A. triste. Inter-
estingly, a recent study in Peru (Mendoza-Uribe and
Chávez-Chorocco 2004) also reported intermediate
forms, similar to the Chilean specimens of the current
study. In fact, one of us (M. B. L.) has examined some
of these Peruvian specimens and has conÞrmed them
to be nearly identical to the present Chilean speci-
mens. Thus, it seems that although typical A. macula-
tum ticks (two stout spurs on metatarsi II-IV) occur
from the United States to the northern part of South
America, and typical A. triste ticks (one spur on meta-
tarsi II-IV) occur in South America (Estrada-Peña et
al. 2005), some intermediate morphological forms
(one stout spur and a less stout spur on metatarsi II-IV)
occur in Peru and northern Chile. Because genetic
analyses have indicated that these three morphotypes
are close related (Fig. 3), further studies are needed
to verify if they represent different taxa or if they are
just geographical races of a single species. In this

Fig. 3. Maximum parsimony phylogenetic tree of 16S rDNA partial sequences (399-bp) of A. maculatum tick group
species. The A. parvitarsum corresponding sequence was used as outgroup. Numbers at nodes are support values derived from
bootstrap, 1,000 replicates. Numbers in brackets are Genbank accession numbers.
222 JOURNAL OF MEDICAL ENTOMOLOGY
regard, it would be very interesting to include in these
analyses, specimens of A. triste recently reported from
Mexico (Guzmán-Cornejo et al. 2006) and the United
States (Mertins et al. 2010), which are considered to
be ÔatypicalÕ localities for A. triste.
Regardless of their taxonomic position, A. macula-
tum and A. triste are of major medical signiÞcance
because they are vectors of the spotted fever group
pathogen, Rickettsia parkeri, in the United States (Pad-
dock et al. 2004), Argentina (Romer et al. 2011), Uru-
guay (Venzal et al. 2004), and Brazil (Silveira et al.
2007). Therefore, the present report of A. triste in
northern Chile alerts us to the possible occurrence of
spotted fever rickettsiosis because of R. parkeri in
northern Chile. Unfortunately, only three specimens
were collected, precluding a more extensive analysis
of rickettsial infection because of R. parkeri. However,
at least one A. triste female was shown to be infected
by Candidatus ÔR. andeanaeÕ, a spotted fever group
agent of unknown pathogenicity that has been re-
ported infecting several Amblyomma species from the
United States to Argentina, including A. maculatum
from the United Sates and Peru (Jiang et al. 2005,
Paddock et al. 2010).
Acknowledgments
We are grateful to A. Marcili (University of São Paulo) for
performing the phylogenetic analysis of the current study.
This work was Þnancially supported by FONDECYT Project
No. 1100809 (Chile) and Fapesp and CNPq (Brazil). Parts if
this work has been facilitated through the “Programa Ibe-
roamericano de Ciencias y Tecnologṍa para el Desarrollo”
(CYTED) to “Red Iberoamericana para la Investigación y
Control de las Enfermedades Rickettsiales” (RIICER).
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Received 18 July 2011; accepted 30 September 2011.