Ticks and Tick-borne Diseases 12 (2021) 101824

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Ticks and Tick-borne Diseases

journal homepage: www.elsevier.com/locate/ttbdis

Original article

Domestic dogs as amplifying hosts of Rickettsia rickettsii for Amblyomma

aureolatum ticks
Lina C. Binder a, *, Alejandro Ramírez-Hernández a, b, Maria Carolina de Azevedo Serpa a,
Jonas Moraes-Filho c, Adriano Pinter d, e, Claudia A. Scinachi d, e, Marcelo B. Labruna a
a
Departamento de Medicina Veterinária Preventiva e Saúde animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil
b
Grupo Parasitología Veterinaria, Universidad Nacional de Colombia, Bogotá D.C., Colombia
c
Mestrado em Medicina e Bem-estar Animal, Doutorado com ênfase em Saúde Única, Universidade Santo Amaro, São Paulo, SP, Brazil
d
Superintendência de Controle de Endemias, São Paulo, SP, Brazil
e
Faculdade de Saúde Pública, Universidade de São Paulo, SP, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Brazilian spotted fever (BSF) is an acute infectious disease caused by the bacterium Rickettsia rickettsii, which is
Rickettsia transmitted by different tick species. Due to deleterious effects caused on ticks, the horizontal transmission of
Spotted fever R. rickettsii through amplifying hosts is crucial for its maintenance in tick populations among BSF-endemic areas.
Tick-borne diseases
The tick Amblyomma aureolatum is the main vector of R. rickettsii in the São Paulo metropolitan area; never­
Transovarial transmission
theless, it is not known which vertebrate could act as an amplifying host for this tick species. Herein, we eval­
uated the potential of domestic dogs – primary hosts for A. aureolatum adults in BSF-endemic areas – to act as
amplifying hosts. For this purpose, A. aureolatum non-infected adults were allowed to feed on two groups of dogs:
the control group (G1), composed of one dog not exposed to R. rickettsii; and, the infected group (G2), composed
of three dogs infected with R. rickettsii via tick parasitism. All G2-dogs became ill, seroconverted to R. rickettsii,
and rickettsial DNA was detected in 87% of the engorged females that fed on them. Transovarial transmission
rate was estimated to be 25% and infected larvae successfully transmitted R. rickettsii to guinea-pigs, confirming
transovarial transmission and vector competence. No rickettsial DNA was detected in individual samples of eggs
or larvae, which precluded the estimation of filial infection rate, but implies that it was low. Our results suggest
that domestic dogs act as amplifying hosts of R. rickettsii for A. aureolatum ticks in BSF-endemic areas in Brazil.

1. Introduction infection and highly efficient in maintaining the infection through

Rocky Mountain spotted fever (RMSF), also known in Brazil as Bra­
zilian spotted fever (BSF), is an acute febrile disease caused by the
bacterium Rickettsia rickettsii. Different tick species have been implicated
as vectors of this bacterium in the Americas. Amblyomma sculptum is the
main vector in Brazil, except for the São Paulo metropolitan area, where
Amblyomma aureolatum is the most important vector (Guedes et al.,
2005; Pinter and Labruna, 2006; Ogrzewalska et al., 2012; Luz et al.,
2019). In the State of São Paulo, A. aureolatum occurs typically in the
eastern region within the Atlantic rainforest mountain domain, where
optimal conditions of high humidity and cool temperatures are provided
throughout the year (Pinter et al., 2004; Barbieri et al., 2015).
Amblyomma aureolatum ticks are highly susceptible to R. rickettsii
transstadial perpetuation and transovarial transmission (Labruna et al.,
2008, 2011). Nevertheless, it was shown that the R. rickettsii infection
induces some reproductive impairment in A. aureolatum females, which
possibly contributes for the low infection rates among A. aureolatum tick
populations in BSF-endemic areas (Pinter and Labruna, 2006; Labruna
et al., 2011; Ogrzewalska et al., 2012). For this reason, it is unlikely that
A. aureolatum populations can sustain R. rickettsii infection through
successive generations solely by vertical transmission; thus, horizontal
transmission through the participation of amplifying vertebrate hosts
seems to be crucial for R. rickettsii maintenance. In order to understand
BSF eco-epidemiology in areas where the incriminated vector is
A. aureolatum, it is important to identify amplifying vertebrate hosts of
R. rickettsii for this tick species.

* Corresponding author at: Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Orlando Marques de Paiva 87, Cidade Uni­
versitária, São Paulo, SP, Brazil, 05508-270.
E-mail address: lina.binder@usp.br (L.C. Binder).

https://doi.org/10.1016/j.ttbdis.2021.101824
Received 12 April 2021; Received in revised form 11 August 2021; Accepted 19 August 2021
Available online 4 September 2021
1877-959X/© 2021 Elsevier GmbH. All rights reserved.
L.C. Binder et al.
Several field studies have shown that A. aureolatum immature stages
use mainly passerine birds as hosts (Arzua et al., 2003; Guglielmone
et al., 2003; Ogrzewalska et al., 2012; Pacheco et al., 2012; Luz et al.,
2017; Ramirez et al., 2020), while the adult stage uses wild canids as
primary hosts, having also successfully adapted to domestic dogs in
areas with high anthropogenic changes (Guglielmone et al., 2003;
Pinter et al., 2004; Barbieri et al., 2014). In the BSF-endemic areas where
A. aureolatum is the main vector, BSF human cases occur mainly close to
forest patches. In such areas, dogs play an important role by carrying
A. aureolatum ticks from these forest patches to human dwellings,
increasing the risk of human exposure to infected ticks (Pinter et al.,
2008; Angerami et al., 2012; Scinachi et al., 2017; Savani et al., 2019).
Although there have been occasional records of A. aureolatum
immature stages on small mammals (Guglielmone et al., 2003), recent
studies failed to detect this tick-host association in areas where larvae
and nymphs of A. aureolatum were found on passerine hosts, indicating
no important role of small mammals as hosts for this tick species (Pinter,
2007; Ogrzewalska et al., 2012). Based on this information, an efficient
amplifying host of R. rickettsii for A. aureolatum should be passerines or
domestic dogs, since they are the most important hosts for the
A. aureolatum immature and adult stages, respectively.
An earlier study demonstrated that two North American passerine
birds (Corvus corax and Pica pica) did not develop rickettsemia after
being inoculated with R. rickettsii (Lundgren et al., 1966). More recently,
Guillen (2014) demonstrated under experimental conditions that pas­
serines of the genus Turdus (natural hosts for A. aureolatum) were not
amplifying hosts of R. rickettsii for A. aureolatum. On the other hand,
previous studies have demonstrated that the domestic dog can act as an
efficient amplifying host of R. rickettsii for Rhipicephalus sanguineus sensu
lato (s.l.) (Piranda et al., 2011) and Dermacentor variabilis (Price, 1954).
Therefore, it is reasonable to consider the hypothesis that domestic dogs
could act as R. rickettsii-amplifying hosts for A. aureolatum in the São
Paulo metropolitan area.
Although rickettsemic dogs were able to infect R. sanguineus s.l. ticks
that fed on them (Piranda et al., 2011), it is not known if A. aureolatum
female ticks that acquire R. rickettsii from a dog could maintain the
infection by transovarial transmission. Several studies with other tick
species demonstrated that R. rickettsii transovarial transmission rates are
usually very low or absent when the primary infection of the tick
occurred in the adult stage; i.e., transovarial transmission was more
efficient when the primary rickettsial infection occurred during the
larval or nymphal acquisition feeding (Burgdorfer and Brinton, 1975;
Piranda et al., 2011; Soares et al., 2012).
Regarding A. aureolatum, females that acquired R. rickettsii during
the larval feeding on rickettsemic guinea pigs transmitted R. rickettsii
transovarially to 100% of their offspring (Labruna et al., 2011). In an
earlier study, when A. aureolatum females acquired R. rickettsii through
feeding on rickettsemic guinea pigs, transovarial transmission did not
occur to the eggs laid during the first two oviposition days, but it did
occur to eggs laid during the subsequent six days (Vallejo-Freire, 1947).
Nevertheless, these two studies evaluated only guinea pigs as the source
of R. rickettsii for A. aureolatum. Noteworthy, one study reported that
rickettsemic dogs were ≈10 times less efficient than rickettsemic guinea
pigs to infect R. sanguineus s.l. larvae (Piranda et al., 2011).
Given the importance of identifying amplifying hosts of R. rickettsii
for A. aureolatum to better understand BSF ecology, the aim of this study
was to evaluate if dogs experimentally infected with R. rickettsii could
serve as an infection source for A. aureolatum adult females and if these
females could transmit the bacterium transovarially to their offspring.
2. Materials and methods
2.1. Dogs and tick colonies
Four adult male Beagle dogs were used in this experiment. The an­
imals were provided by a commercial breeder and kept in individual
2
Ticks and Tick-borne Diseases 12 (2021) 101824
cages (4 × 2 m), being fed daily with commercial dog food and water ad
libitum. Before starting the experiment, dogs were clinically healthy and
their serum samples were tested by indirect immunofluorescence assay
(IFA) as described elsewhere (Labruna et al., 2007). All serum samples
showed to be non-reactive (serum dilution 1:64) to crude antigens of
Rickettsia amblyommatis, Rickettsia bellii, Rickettsia parkeri, Rickettsia
rhipicephali and R. rickettsii.
For canine infestations with ticks, two separated cotton sleeve
chambers were glued onto the shaved dorsum of each dog one day
before the first tick infestation. These chambers were labelled as
chamber A (cranial position on the dog`s dorsum) and chamber P
(caudal position on the dog`s dorsum). Dogs were assigned to two
experimental groups: the control group (G1), composed by only one dog
(dog no. 1); and the infected group (G2), composed by three dogs (dogs
no. 2, 3 and 4).
All adult ticks used in the canine infestations derived from two tick
colonies (the infected and the non-infected colonies) that were estab­
lished in the laboratory from uninfected engorged females collected
from dogs in a BSF-endemic area (Mogi das Cruzes municipality, São
Paulo metropolitan area). Adult ticks from the infected colony were
experimentally infected during the previous larval stage by feeding on
rickettsemic guinea pigs that were intraperitoneally inoculated with a
homogenate of R. rickettsii strain Taiaçu-infected guinea pig organs, as
previously described (Labruna et al., 2008; 2011). This rickettsial strain
was originally isolated from an A. aureolatum tick collected on a dog in
the same municipality where the progenitors of our tick colony were
collected (Pinter and Labruna, 2006). These progenitors were shown to
be free of rickettsial infection by testing the females at the end of
oviposition, and a sample of their respective eggs, which did not yield
rickettsial DNA by the real-time PCR assay described below. Before
canine infestations, a sample of 21 unfed ticks (17 nymphs and four
adults) of the infected colony was tested individually for rickettsial DNA
by PCR (protocols described below). Since all 21 ticks yielded rickettsial
DNA, the R. rickettsii-infection rate of the infected colony was compat­
ible with 100%.
2.2. Canine infestations with A. aureolatum adults
The first tick infestation on dogs was performed on day 0 (zero),
when G1 dog was infested with ten non-infected males in chamber A,
and G2 dogs were infested each with ten R. rickettsii–infected males in
chamber A. On day 2, each dog from G1 or G2 group was infested by
twelve non-infected females in each of the two chambers (A and P) and
by ten non-infected males in chamber P. On day 7, five additional non-
infected females were allowed to feed in chamber P in G1 and G2 dogs,
and, finally, on day 9 these four dogs were infested with four additional
non-infected females on both chambers (A and P). A schematic design of
these canine infestations is presented in Fig. 1.
Both feeding chambers were opened daily and engorged females
were collected and transported to an incubator (23 ± 1 ◦ C and 95–100%
RH) for oviposition. Engorged females were individually weighed in an
electronic balance (precision of 0.0001 g) the day they detached from
host. Additionally, the total egg mass laid by each female was weighed at
the end of oviposition and the conversion efficiency index (CEI = egg
mass weight/engorged female weight x 100) was determined following
Drummond and Whetstone (1970).
Dogs were monitored daily for rectal temperature and clinical ab­
normalities until 26 days following the first infestation. In addition,
blood samples were collected every two days within the 26-day period
and divided into two aliquots: one for DNA extraction and a second to
obtain serum for IFA tests. As one dog died during the experiment, tissue
samples were collected from different organs (brain, kidney, liver, lung
and spleen) and stored at − 20 ºC until molecular analyses.
L.C. Binder et al.
Fig. 1. Experimental infestations in dogs with Rickettsia rickettsii-infected and non-infected Amblyomma aureolatum adult ticks. Control group (G1) composed of one
dog; infected group (G2) composed of three dogs. Each dog had two infestation chambers (A and P) glued onto its dorsum.
2.3. Serological analyses
All canine serum samples were tested by IFA using R. rickettsii (strain
Taiaçu) crude antigens and a fluorescein isothiocyanate-labeled rabbit
anti-dog IgG (Sigma Diagnostics, St. Louis, MO). Sera samples were
initially tested at the 1:64 serum dilution as cut-off, and those reacting at
1:64 were further diluted in twofold increments to determine the
endpoint titer, as previously described (Labruna et al., 2007). In each
slide, canine non-reactive (negative control) and reactive (positive
control) sera from a previous study (Piranda et al., 2008) were tested at
the 1:64 dilution.
2.4. Molecular analyses
Engorged females at the end of oviposition as well as one pool of eggs
and one pool of unfed larvae from each progeny were submitted to DNA
extraction by the guanidine isothiocyanate-phenol protocol (Sangioni
et al., 2005). Females were processed individually, while eggs and larvae
were processed in pools of 50 and 20 individuals, respectively. When an
egg or larval pool showed to contain rickettsial DNA, 20 eggs and larvae
from the respective batch were individually tested in order to estimate
filial infection rate. DNA from canine blood and tissue samples was
extracted using the DNeasy Blood and Tissue kit (Qiagen Inc., Valencia,
CA, USA) following the manufacturer`s protocols.
All DNA samples were initially tested by a real-time PCR assay that
amplifies a 147-bp fragment of the citrate synthase gene (gltA) of Rick­
ettsia spp. using the primers CS-5 (Guedes et al., 2005) and CS-6 (Lab­
runa et al., 2004), and an internal fluorogenic probe (6-FAM d, BHQ- 1)
3
Ticks and Tick-borne Diseases 12 (2021) 101824
(Integrated DNA Technologies, San Diego, CA), following reagents and
cycling conditions reported by Soares et al. (2012). Each reaction
included positive (DNA of Rickettsia vini cultivated in Vero cells) and
negative control (molecular-grade water) samples. Real-time PCR assays
were performed in a 7500 Real-time PCR System apparatus (Applied
BioSystems, Foster City, CA, USA).
DNA samples considered positive by the gltA real-time PCR protocol
were further tested by a conventional PCR targeting a 632-bp fragment
of the 190-kDa outer membrane protein gene (ompA), using the primers
Rr190.70 (Regnery et al., 1991) and Rr190.701 (Roux et al., 1996). Each
reaction included positive and negative control samples, as mentioned
above. In the Results section, samples containing rickettsial DNA were
those that yielded PCR-positive results by both real-time PCR (cycle
threshold < 35) and ompA-conventional PCR.
As quality control for probable amplification inhibition and for the
DNA extraction method, all samples that tested negative for rickettsial
DNA by the real-time PCR were retested. Tick samples were tested by a
conventional PCR targeting a 460 bp-fragment of the tick mitochondrial
16S rRNA gene, as described (Mangold et al., 1998), while blood and
tissue samples were tested using CYTB1 and CYTB2 primers, designed to
amplify a 359 bp-fragment of the mitochondrion cytochrome b gene (cyt
b) of vertebrates (Steuber et al., 2005). Samples that yielded negative
results were excluded from further analyses.
Amplicons of the ompA-PCR of eight tick DNA samples (seven fe­
males and one egg pool) were selected for rickettsial DNA sequencing.
PCR products were treated with ExoSap (USB, Cleveland, OH, United
States) and sequenced in an ABI automated sequencer (Applied Bio­
systems/Thermo Fisher Scientific, model ABI 3500 Genetic Analyzer,
L.C. Binder et al. Ticks and Tick-borne Diseases 12 (2021) 101824

Foster City, CA, USA). Generated sequences were further compared and
aligned using the BLAST tool (Altschul et al., 1990) to confirm the
Rickettsia identity.
In order to assure that dogs had not been infected by other known
canine tick-borne pathogens during the infestations, canine blood sam­
ples were also tested by two other real-time PCR protocols: (i) for Ehr­
lichia canis (gene dsb) detection (Doyle et al., 2005); and (ii) for Rangelia
vitalii (gene hsp70) detection (Soares et al., 2018). For this purpose, only
samples collected during the febrile period of dogs were tested.
2.5. Guinea pig infestations
To confirm successful transovarial transmission of R. rickettsii, larvae
derived from infected engorged females were allowed to feed on 12 tick-
naïve guinea pigs. For this purpose, 50 – 500 larvae (depending on the
availability of ticks) from each of 12 infected females were released in a
cotton sleeve chamber that was glued onto the shaved dorsum of a
guinea pig. Blood samples were collected intracardiacally under anes­
thesia from each guinea pig on the infestation day, and 21 days after.
Sera were separated by centrifugation and stored at − 20 ◦ C until tested
by IFA [as described above, except that a rabbit anti-guinea pig IgG
conjugate (Sigma Diagnostics, St. Louis, MO) was used]. Guinea pigs
were daily monitored for rectal temperature during the infestation
period (21 days) and were considered febrile when rectal temperatures
higher than 39.5 ◦ C were registered for two or more consecutive days,
following previous studies (Labruna et al., 2011; Saraiva et al., 2014).
Furthermore, feeding chambers were opened daily and naturally de­
tached engorged larvae were collected.
2.6. Statistical analyses
Reproductive data of engorged females were compared between fe­
males fed on G1 and G2 dog groups. Oviposition success was compared
using chi-square test, while engorged female weight, egg mass weight
and CEI were compared by using the t-test after the Kolmogorov-
Smirnov normality test. A 95% confidence level was assumed, and all
tests were performed on R (R Core team 2019).
2.7. Ethics statement
the cause of this late temperature increase. As expected, dog no. 1 (non-
infected G1 group) showed no clinical abnormalities during the study
period.
Dog no. 3, which was euthanized 20 DPI, was submitted to necropsy.
On gross examination hepatomegaly and splenomegaly were observed,
as well as disseminated lesions suggestive of vascular alterations
(congestion and hemorrhage) in lungs, brain and skin (Supplementary
Fig. S2).
All G2 dogs seroconverted within 7–11 DPI and reciprocal titers of
IgG antibodies against R. rickettsii reached 8192 (dogs no. 3 and 4) and
65,636 (dog no. 2) by 19–25 DPI (Supplementary Fig. S3). As expected,
dog no. 1 (non-infected group) remained seronegative to R. rickettsii at
the 1:64 serum dilution throughout the study.
Rickettsial DNA was not detected in any of the canine blood samples
nor from the tissue samples from dog no. 3 by real-time PCR. Rangelia
vitalii and E. canis DNA were neither detected, excluding the possibility
of coinfection by one of these pathogens. Vertebrate mitochondrion
DNA was successfully amplified in all samples by conventional PCR,
confirming the suitability of the DNA extraction.
3.2. Ticks
Overall, 99 engorged females were recovered from the four dogs, and
69 of them successfully oviposited. Between G1 and G2 dogs, there were
no statistical differences between female engorged weight, oviposition
success, egg mass weight or CEI (Table 1).
Rickettsial DNA was detected by PCR in 87% (49/56) of the
engorged females fed on G2 dogs; additionally, 4% (2/49) of their
respective egg pools yielded rickettsial DNA, indicating that at least 4%
of the infected females were able to transmit R. rickettsii to their
offspring. Nevertheless, rickettsial DNA was not detected in any of 47
larval pools derived from infected females (Table 2), neither from in­
dividual eggs nor larvae from the only two engorged females that yiel­
ded PCR-positive egg pools.
Through DNA sequencing, amplicons of the ompA-PCR from eight
samples (seven engorged females and one egg pool) generated a
consensus sequence 100% identical to the ompA partial sequence of
R. rickettsii available in GenBank (NC016913).

All animal procedures of the present study were authorized by the 3.3. Guinea pig infestations
Ethic Committee on Animal Use of the Faculty of Veterinary Medicine
and Animal Science (University of São Paulo) (CEUA No. 1878230418). Larval offspring of 12 females were used for guinea pig infestations.
All these 12 females fed on dogs during their febrile period and were
3. Results shown to contain rickettsial DNA at the end of oviposition. Nevertheless,
only two of them yielded rickettsial DNA in their eggs, whereas the
3.1. Canine infection
Table 1
All three dogs of the infected group (G2) had fever (rectal tempera­ Reproductive data of Amblyomma aureolatum females after feeding on non-
ture >39.5ºC) with onset at 8 or 9 days post-infestation (DPI), which infected dogs (control group) and Rickettsia rickettsii-infected dogs (infected
lasted for two to five days (Supplementary Fig. S1). The highest tem­ group).
perature was observed in dog no. 3 (40.5 ºC), which also presented the Dogs No. females that Engorged Egg CEI (egg mass
longest febrile period (5 days). All G2 dogs presented hyporexia and Group No. oviposited/ no. female mass weight
recovered weight weight /engorged
lethargy within the febrile period. Diarrhea and ocular discharge were engorged females (mg)a (mg)a female weight
observed in dogs no. 2 and 3, lasting for four days. Dogs no. 2 and 4 (% oviposition × 100)a
recovered completely (15 and 10 DPI, respectively), but dog no. 3 success)
showed rectal temperature above 39.5 ºC at the 17th DPI, preceded by Control 1 10/20 (50) 785.1 458.4 55.6 [15.5]
three days of normal temperature. In the absence of clinical improve­ [245.2] [189.4]
ment in dog no. 3, antibiotic treatment was initiated 17 DPI (doxycy­ Infected 2 23/31 (74) 989.5 463.4 45.0 [24.6]
cline 5 mg/kg orally twice a day). The treatment was not effective, and [284.0] [265.8]
3 18/21 (86) 823.2 456.0 57.4 [14.4]
at 19 DPI, dog no. 3 was not able to stand, presented head tremors, limbs [188.7] [166.4]
stiffness and nystagmus. Dog no. 3 was euthanized 20 DPI by intrave­ 4 18/27 (67) 838.7 422.5 44.6 [23.7]
nous administration of pentobarbital sodium. Tissue samples were [282.9] [221.7]
collected and stored at − 20ºC for molecular analyses. Surprisingly, be­ 2, 59/79 (75) 895.4 448.2 48.5 [22.2]
3, 4 [270.5] [222.8]
tween 23 and 25 DPI, dogs no. 2 and 4 presented high rectal tempera­
tures again, with no other clinical signs. We were not able to elucidate a
Mean [standard deviation].

4
L.C. Binder et al. Ticks and Tick-borne Diseases 12 (2021) 101824

Table 2
Rickettsia rickettsii-DNA detection in Amblyomma aureolatum females and in their respective eggs and larvae according to the dog and feeding chamber where the
females fed on, as well as results of IFA of guinea-pigs infested with larval offspring of the infected females.
Dogs PCR on ticks:No. infected/No. tested (% infection) Guinea pigs: No. that seroconverted / No. infested with larvae(%
Group No. Feeding Females after Egg pools Larval pools seroconversion)a
chamber oviposition

Control 1 A 0/4 (0) 0/3 (0) 0/4 (0) Not done

P 0/6 (0) 0/6 (0) 0/6 (0) Not done
A+P 0/10 (0) 0/9 (0) 0/10 (0) Not done
Infected 2 A 8/9 (89) 0/8 (0) 0/6 (0) Not done
P 13/14 (93) 1/13 (8) 0/13 (0) 1/1 (100)
3 A 8/10 (80) 0/8 (0) 0/8 (0) 0/1 (0)
P 6/8 (75) 0/6 (0) 0/6 (0) 0/2 (0)
4 A 6/6 (100) 1/6 (17) 0/6 (0) 1/3 (33)
P 8/9 (89) 0/8 (0) 0/8 (0) 1/5 (20)
2,3,4 A 22/25 (88) 1/22 (4) 0/20 (0) 1/4 (25)
P 27/31 (87) 1/27 (4) 0/27 (0) 2/8 (25)
A+P 49/56 (87) 2/49 (4) 0/47 (0) 3/12 (25)
a
Each of the 12 guinea-pigs was infested with larvae from a single engorged female of the infected group, including two females that yielded Rickettsia-infected egg
pools by PCR and 10 females that did not yielded Rickettsia-infected egg pools. Seroconversion was demonstrated by endpoint titer ≥1024 to Rickettsia rickettsii at 21
days post larval infestation on guinea pigs, which were all non-reactive (titer <64) at day 0.

remaining 10 were randomly selected, as their egg and larval pools
yielded no rickettsial DNA by PCR. Each of 12 tick-naïve guinea-pigs was
infested with 50 – 500 larvae derived from a single infected female; this
variable number of larvae relied on the variable hatchability of the egg
masses.
All 12 guinea pigs were serologically non-reactive to R. rickettsii (titer
< 64) at the infestation day, and three out of them seroconverted to
R. rickettsii 21 days after infestation (endpoint titers ≥1024). These same
three animals manifested fever (rectal temperature >39.5 ºC) starting
between 8 and 10 days after infestation, and lasting for 3 to 5 days
(Table 3). The remaining nine guinea pigs remained afebrile during the
21-day observation period.
Among the three febrile guinea pigs that seroconverted, only one
(guinea pig code: 2.16) was infested with larvae derived from egg batch
that yielded Rickettsia-infected egg pool; the other two (4.5 and 4.11)
were infested with larvae derived from egg batches that yielded no
rickettsial DNA in egg or larval pools (Table 3). On the other hand, one
guinea pig (4.3) was infested with larvae from an egg batch that yielded
a Rickettsia-infected egg pool, but this animal neither manifested fever
nor seroconverted. Noteworthy, this animal was infested with only ~50
larvae that hatched from a small egg batch (egg mass: 59.5 mg) with low
hatchability.
4. Discussion
This is the first study to assess the competence of domestic dogs to act
as amplifying hosts of R. rickettsii for A. aureolatum ticks. We were able to
demonstrate that all dogs infected with R. rickettsii via tick feeding
developed typical illness, and that a fraction of the rickettsemic dog-fed
A. aureolatum females was able to maintain R. rickettsii through trans­
ovarial transmission. The first mathematical model describing the
R. rickettsii maintenance cycle addressed the importance of the ampli­
fication of the bacterium in subadult tick hosts (Cooksey et al., 1990).
The present study is the first to propose that, differently from other
R. rickettsii-tick vectors, in A. aureolatum the bacterial amplification
process and horizontal propagation of R. rickettsii among ticks may be
utterly supported through the adult tick-acquisition feeding, and then,
through transovarial transmission.
All infected dogs showed clinical manifestations compatible with
those observed in natural cases of BSF and in other studies of canine
experimental infection with R. rickettsii (Gasser et al., 2001; Piranda
et al., 2008; Labruna et al., 2009; Levin et al., 2014), being marked by
fever, lethargy, hyporexia, gastrointestinal disorders, ocular discharge
and neurological manifestations. Although all infected dogs were of the
same breed, were of similar ages, and were exposed to the same number
of infected ticks, the clinical manifestations observed were quite
different in terms of severity. While dog no. 4 presented only moderate
fever, dog no. 3 had severe neurological manifestations becoming
moribund within 2.5 weeks postinfection.
Although the dogs of the present study were infected with the same
R. rickettsii strain used in the study by Piranda et al. (2008), the present
clinical pictures were notably more severe than the ones observed by

Table 3
Clinical data of 12 guinea pigs that were infested with Amblyomma aureolatum larvae derived from Rickettsia rickettsii-infected females, with data of the origin of the
larvae and results of PCR for rickettsiae on egg pools from the egg batches that originated the larvae.
Guinea pig Origin of the larvae PCR for rickettsiae on egg Guinea pig datab
code Dog Feeding Female detachment pool Fever onset Maximal temperature ( IFA endpoint
No. chamber daya (DPI) ◦
C) titer

2.16 2 P 17 positive 8 40.4 ≥1024

3.10 3 A 18 negative no fever 38.7 <64
3.2 3 P 12 negative no fever 39.0 <64
3.6 3 P 14 negative no fever 38.9 <64
4.3 4 A 11 positive no fever 38.6 <64
4.5 4 A 12 negative 9 40.6 ≥1024
4.10 4 A 17 negative no fever 38.2 <64
4.5 4 P 13 negative no fever 38.9 <64
4.9 4 P 14 negative no fever 38.8 <64
4.10 4 P 14 negative no fever 38.8 <64
4.11 4 P 14 negative 10 40.7 ≥1024
4.14 4 P 18 negative no fever 38.4 <64
a
Refers to the day post infestation of the dog with R. rickettsii-infected A. aureolatum males.
b
DPI: days post infestation with larvae; IFA: immunofluorescence assay with R. rickettsii antigen.

5
L.C. Binder et al.
Piranda et al. (2008) in dogs infected via tick parasitism. Levin et al.
(2014) observed that older dogs had more severe clinical manifestations
than younger dogs infected with the same strain of R. rickettsii. Thus, the
difference in clinical conditions between the two studies could be
associated with the age of the dogs, since Piranda et al. (2008) used
six-month-old dogs, while the animals of the present study were 2–3
years old.
Doxycycline is considered the drug of choice for BSF treatment in
dogs, with remission of symptoms observed within 48 h after beginning
of treatment (Breitschwerdt et al., 1997; 1999). In our study, treatment
with doxycycline did not prevent disease progression and the dog
evolved to a severe neurological picture and had to be euthanized. Delay
in starting treatment (17 DPI) was probably the cause of unsatisfactory
response to treatment, since even in dogs presenting neurological signs,
the administration of doxycycline up to 14 days after infection was re­
ported to fully revert the symptoms (Gasser et al., 2001; Labruna et al.,
2009; Levin et al., 2014). Gastrointestinal disorders probably due to
vascular injury may also have contributed to treatment failure, since the
drug was administered orally. Interestingly, R. rickettsii DNA was not
detected in any of the tested tissue samples, which was not completely
unexpected, as the dog was under a doxycycline therapy that had
initiated two days prior to its death. The late beginning of treatment was
presumably able to reduce bacterial load, but as vascular injury was by
that time disseminated (as shown by hemorrhagic lesions on internal
organs), it was not able to prevent disease progression. Anyhow, the
postmortem pathological findings in dog no. 3 are consistent with
R. rickettsii infection in dogs (Kidd and Breitschwerdt, 2013).
Dogs no. 2 and 4, which survived the infection, had complete
remission of symptoms without any type of sequelae. The cause of fever
relapse experienced by both animals 23 days after infestation remains
unclear. Interestingly, Breitschwerdt et al. (1999) noticed in their study
that all groups of dogs experimentally infected with R. rickettsii also
experienced a relapse of fever of unclear cause about ten days after the
first febrile period. Furthermore, Souza et al. (2009) detected the pres­
ence of viable R. rickettsii bacteria in the blood of experimentally
infected capybaras about 15 days after the first bacteremia. One of the
dogs experimentally infected in the study by Levin et al. (2014) pre­
sented relapse approximately 30 days after the first febrile period. Based
on these findings, it is reasonable to assume that relapses may not be
uncommon in dogs infected with R. rickettsii, and their possible causes
deserve to be further investigated.
G2 dogs showed high titers for R. rickettsii a few days after the
infestation, showing that all of them had been exposed to the bacterium,
in contrast to the G1 dog. In addition, the three G2 dogs developed
rickettsemia that possibly infected new ticks, since R. rickettsii DNA was
detected in more than 87% of the recovered engorged females. Rick­
ettsial DNA was detected in females that fed on either chamber A (where
infected males and uninfected females fed together) or chamber P
(where uninfected females fed physically separated from infected
males), confirming R. rickettsii systemic transmission. Similar results
were obtained by Piranda et al. (2011), who detected R. rickettsii in
100% of R. sanguineus s.l. females fed on infected dogs. These authors
were also able to detect rickettsial DNA in blood samples (Piranda et al.,
2008), in contrast to our study, in which rickettsial DNA was not
detected in any of the blood samples.
In other experimental studies in which dogs were infected with
R. rickettsii by tick parasitism, rickettsial DNA was amplified from blood
samples obtained over a period of one to nine days (Piranda et al., 2008;
Levin et al., 2014). In our study, blood samples were collected on
alternate days, which reduced the sensitivity of our detection. In addi­
tion, Levin et al. (2014) noted that older dogs had shorter periods of
bacteremia detectable via PCR (1 to 3 days) than younger dogs (7 to 9
days). This could indicate that the dogs in this study, all > 2 years old,
could have detectable levels of R. rickettsii for a shorter period of time.
Finally, studies that evaluated experimental infection with
R. rickettsii in capybaras (Hydrochoerus hydrochaeris) and opossums
6
Ticks and Tick-borne Diseases 12 (2021) 101824
(Didelphis aurita) also failed to detect the presence of rickettsial DNA in
the blood of most infected animals known to be rickettsemic (Horta
et al., 2009; Souza et al., 2009; Ramírez-Hernández et al., 2020). The
number of circulating R. rickettsii organisms is expected to be low in
infected vertebrates, since the bacterium infects endothelial cells and,
therefore, these results are not entirely unexpected.
Labruna et al. (2011) found that R. rickettsii has deleterious effects on
engorged females of A. aureolatum, since they recorded smaller egg
masses oviposited by infected females and a higher mortality among
them when compared to non-infected females. In our study, however, no
statistically significant differences were observed for the measured
biological parameters between females from the infected group and
those from the control group.
Burgdorfer and Brinton (1975) observed that deleterious effects
caused by R. rickettsii in engorged females were related to the degree of
rickettsial development in ovarian tissues and that the extent of rick­
ettsial reproduction in ovarian tissues depended primarily on the time
elapsed from female infection until the beginning of oviposition. Thus,
females infected as larvae or nymphs, as occurred in the experiment by
Labruna et al. (2011), would present a generalized massive infection by
R. rickettsii in the ovarian tissue, leading to a less efficient reproductive
performance of these individuals. On the other hand, females primarily
infected in the adult phase, as in the present study, would present a
much less extensive ovarian infection, which could explain the absence
of deleterious effects observed in the reproductive performance of
infected females when compared to control group females.
Differently from Labruna et al. (2011), who detected rickettsial DNA
in 100% of egg pools originated from infected females, we found less
than 5% of the egg pools tested by PCR to be infected with R. rickettsii.
This low proportion of infected egg pools is also probably related to
female late infection. Oocyte development in the tick ovary is an asyn­
chronous process, in which the vitelline membrane, which prevents
penetration of R. rickettsii into oocytes, is not formed in all oocytes at the
same time (Burgdorfer and Brinton, 1975). In females infected as larvae
or nymphs, bacteria are able to massively infect oocytes before the
formation of the vitelline membrane. In contrast, when females are
primarily infected as adults, rickettsiae are only able to infect eggs that
are laid during later phases of oviposition, before the complete devel­
opment of the vitelline membrane in the ovary (Vallejo-Freire, 1947;
Burgdorfer and Brinton, 1975).
Due to the asynchronous process of tick oogenesis and R. rickettsii
multiplication in the ovaries, female ticks infected as adults produce
heterogenous egg batches regarding R. rickettsii infection. The hetero­
geneity of the egg batch makes it more difficult to assess transovarial
transmission rate (proportion of infected females giving rise to at least
one infected egg) and filial infection rate (proportion of infected eggs
obtained from an infected female) from females infected as adults, since
infected eggs are not evenly distributed in the egg mass, what makes the
estimate from just one or a few samples highly inaccurate. This state­
ment is in agreement with the early study of Vallejo-Freire (1947), in
which transovarial transmission of R. rickettsii in A. aureolatum was
detected only after the first two oviposition days for females that had
acquired infection through feeding on rickettsemic guinea pigs.
In our study, it was possible to detect rickettsial DNA in only two egg
pools, which would lead us to believe that transovarial transmission rate
was only 4% (2/48). However, when infestations on guinea pigs were
performed, it was observed that larvae originated from three out of
twelve females were infected with R. rickettsii, showing that transovarial
transmission rate was 25%. The fact that R. rickettsii DNA was not
detected in any of the tested larval pools nor in any individually eggs or
larvae made it impossible for us to estimate filial infection rate, but it
indicates that this proportion is probably low. This result is corroborated
by previous studies, which reported a partial rickettsial transovarian
transmission when female ticks were primarily infected as adults
(Burgdorfer and Brinton, 1975; Piranda et al., 2011; Soares et al., 2012).
In this study we demonstrated that rickettsemic dog-fed
L.C. Binder et al.
A. aureolatum females were able to transmit R. rickettsii efficiently to part
of their progeny (transovarial transmission), showing that the horizontal
transmission of R. rickettsii through this vertebrate host could start new
lineages of infected ticks, amplifying the infection in the A. aureolatum
population. Although not evaluated, it is quite possible that infected
adult females derived from the infected larvae of the present study
would generate nearly 100% transovarial transmission and filial infec­
tion rate, as it has been demonstrated for females after R. rickettsii-
acquisition feeding as larvae (Labruna et al., 2011).
Based on the results of the present study, couple with the fact that
immature stages of A. aureolatum feed primarily on passerine hosts
among BSF-endemic areas (Pinter, 2007; Ogrzewalska et al., 2012),
domestic dogs are likely to act as the main amplifying hosts of R. rickettsii
for A. aureolatum ticks in BSF-endemic areas of the São Paulo metro­
politan area. Indeed, such statement needs to be confirmed by further
studies, including long-term epidemiological evaluations.
CRediT authorship contribution statement
Lina C. Binder: Conceptualization, Methodology, Resources, Formal
analysis, Writing – original draft, Writing – review & editing. Alejandro
Ramírez-Hernández: Investigation, Writing – review & editing. Maria
Carolina de Azevedo Serpa: Investigation, Writing – review & editing.
Jonas Moraes-Filho: Investigation, Writing – review & editing.
Adriano Pinter: Investigation, Resources, Writing – review & editing.
Claudia A. Scinachi: Investigation, Writing – review & editing. Mar­
celo B. Labruna: Conceptualization, Methodology, Resources, Formal
analysis, Writing – review & editing, Supervision, Project administra­
tion, Funding acquisition.
Acknowledgements
This work is dedicated to Francisco Uchoa, who helped us enor­
mously during the experiment and unfortunately passed away in 2020.
We are very grateful to SUCEN Mogi Guaçu for the logistic support
during the experiment and to Laboratórios Ecolyzer Ltda for the dona­
tion of dogs for the present study. This study was supported by the São
Paulo Research Foundation (FAPESP grant no. 2018/01596-8); Con­
selho Nacional de Desenvolvimento Científico e Tecnológico (CNPq
grant no. 425078/2016-7); and Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior – Brasil (CAPES Finance code 001).
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.ttbdis.2021.101824.
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