Medical and Veterinary Entomology (2015), doi: 10.1111/mve.

12118

The role of Rhipicephalus sanguineus sensu lato saliva

in the dissemination of Rickettsia conorii in C3H/HeJ
mice
N. M I L H A N O 1 , T. B. S A I T O 2, , J. B E C H E L L I 2, , R. F A N G 2 ,
M. V I L H E N A 3 , R. DE S O U S A 1 and D. H. W A L K E R 2
1

Centre for the Study of Vectors and Infectious Diseases Dr Francisco Cambournac, National Institute of Health Dr Ricardo Jorge,
guas de Moura, Portugal , 2 Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, University of Texas
Medical Branch, Galveston, TX, U.S.A. and 3 Department of Veterinary Medicine, University of vora, vora, Portugal

Abstract. Animal models have been developed for the study of rickettsial pathogenesis. However, to understand what occurs during the natural route of rickettsial transmission via the tick bite, the role of tick saliva should be considered in these models. To
address this, we analysed the role of tick saliva in the transmission of Rickettsia conorii
(Rickettsiales: Rickettsiaceae) in a murine host by intradermally (i.d.) inoculating two
groups of susceptible C3H/HeJ mice with this Rickettsia, and infesting one group with
nymphal Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) ticks. Quantification
of bacterial loads and mRNA levels of interleukin-1 (IL-1), IL-10 and NF-B was performed in C3H/HeJ lung samples by real-time quantitative polymerase chain reaction
(PCR) and real-time reverse transcriptase PCR, respectively. Lung histology was examined to evaluate the pathological manifestations of infection. No statistically significant
difference in bacterial load in the lungs of mice was observed between these two groups;
however, a statistically significant difference was observed in levels of IL-1 and NF-B,
both of which were higher in the group inoculated with rickettsiae but not infected with
ticks. Lung histology in both groups of animals revealed infiltration of inflammatory
cells. Overall, this study showed that i.d. inoculation of R. conorii caused infection in
the lungs of C3H/HeJ mice and tick saliva inhibited proinflammatory effects.
Rhipicephalus sanguineus sensu lato, Rickettsia conorii, intradermal
inoculation, real-time PCR, tick saliva.

Key words.

Introduction
Ticks are obligately haematophagous ectoparasites and their
saliva plays a fundamental role in thwarting the host immune
system during their prolonged feeding period, thus facilitating the transmission of pathogens (Wikel, 2013; Liu & Bonnet, 2014). Pharmacologically active components contained in
tick saliva inhibit haemostasis, reduce inflammation, and suppress or modulate host immune responses (Ribeiro, 1987, 1995;

Kazimrov & Stibraniova, 2013). This ability to circumvent

immunity is essential to the ticks success in feeding and has
been shown to enhance the efficiency of pathogen transmission (Chen et al., 2012; Lieskovska & Kopecky, 2012). A prime
example of this capacity is demonstrated in the brown dog
tick Rhipicephalus sanguineus sensu lato, which is the most
widely distributed tick globally and amongst the most important vectors of pathogenic agents to both humans and animals
(Dantas-Torres, 2010; Otranto et al., 2014). Rhipicephalus san-

Correspondence: Natacha Milhano, Centro de Estudos de Vectores e Doenas Infecciosas Dr Francisco Cambournac, Instituto Nacional
de Sade Dr Ricardo Jorge, Avenida da Liberdade, 5, 2965-575 guas de Moura, Portugal. Tel.: + 351 26 593 8306; Fax: + 351 26 591 2155;
E-mail: natachamil@yahoo.com.br
These

authors contributed equally to this study.

2015 The Royal Entomological Society

2 N. Milhano et al.
guineus s.l. is the vector of Rickettsia conorii, responsible for
Mediterranean spotted fever (MSF), an endemic rickettsial disease in the Mediterranean basin, India, Russia and Africa. Many
studies to elucidate rickettsial pathogenesis in the host have
been undertaken, based on the development of three endothelial target murine models of rickettsioses, namely C3H/HeN
mice for the study of R. conorii (Walker et al., 1994) and Rickettsia typhi (Walker et al., 2000), and BALB/c mice or C57Bl/6
mice for Rickettsia australis (Feng et al., 1993; Walker et al.,
2001). However, there is still a gap in our knowledge of the
role of tick saliva in the natural route of rickettsial transmission to the host. Furthermore, the models developed thus far
have employed the intravenous (i.v.) route of infection, whereas
intradermal (i.d.) inoculation of the skin, which would more
closely resemble the natural route of infection, has yet to be
described. We performed preliminary experiments to evaluate
the i.d. route of infection in C3H/HeJ mice and observed consistent infection in the lungs. In order to address the limitations
of earlier models, in the present study we used non-infested
and Rh. sanguineus s.l. nymph-infested R. conorii i.d. inoculated
C3H/HeJ mice, a strain shown to represent a more susceptible
model for the study of R. conorii pathogenesis (Eisemann et al.,
1984). The main objective of this study was to analyse the effects
of Rh. sanguineus s.l. saliva on the dissemination of R. conorii
in the host, using an i.d. route of rickettsial inoculation in order
to mimic the natural tick bite route of inoculation.

mice were each inoculated i.d. with 1 107 copies of R. conorii

Israeli spotted fever (ISF) strain (RC/tick infestation group) and
the three controls were inoculated i.d. with phosphate-buffered
saline (PBS). In the other group, five mice were inoculated i.d.
with 1 107 copies of R. conorii ISF strain (RC group) and the
other three (controls) with PBS. The rickettsial inoculation site
was in the region of the capsule containing the ticks. The animals
were monitored daily for signs of disease and killed at 5 days
post-inoculation, a time-point previously shown to correspond
to a rickettsial peak (Walker et al., 1994), which also coincided
with the engorgement of the majority of the nymphs. Lung
samples were collected and processed for analysis of rickettsial
loads, cytokine and transcriptional factor levels and histology.

Tick infestation
A capsule made from a cut-off 2-mL microtube (Sarstedt AG
& Co., Nmbrecht, Germany) was attached to the shaved back of
each sedated mouse (24% isoflurane inhalation) using Kamar
adhesive (Kamar Products, Inc., Zionville, IN, U.S.A.), which is
routinely used in livestock for adhesion of Kamar HeatMount
Detectors (Kamar Products, Inc.). On the following day, 10
Rh. sanguineus s.l. nymphs were counted and introduced into
the capsule on each mouse, and allowed to feed to repletion.

Rickettsial preparation

Materials and methods

Mice and ticks
All experiments were performed using C3H/HeJ mice (Jackson Laboratory, Bay Harbor, ME, U.S.A.) aged 46 weeks,
housed in an animal biosafety level 3 facility at the University
of Texas Medical Branch (UTMB).
Rhipicephalus sanguineus s.l. nymphs were obtained from the
Oklahoma State University Tick Rearing Facility (Stillwater,
OK, U.S.A.). The ticks were placed in small glass vials sealed
with a mesh and plastic lid pierced with holes in order to allow
for air exchange, and kept in desiccators at room temperature
and approximately 90% relative humidity.
All experiments and procedures were performed according to
protocols approved by the Institutional Animal Care and Use
Committee at UTMB. The animal care and use programme at
UTMB conducts reviews involving animals in accordance with
the 2011 Guide for the Care and Use of Laboratory Animals
(National Research Council, National Academies, Washington,
DC, U.S.A.). The Animal Resource Center at UTMB provides
a comprehensive programme of veterinary care, animal husbandry, technical advice, assistance and facilities.

Experimental design
C3H/HeJ mice were divided into two groups, each containing
five experimental and three control animals. In one group of
animals, eight mice were infested with 10 Rh. sanguineus s.l.
nymphs each; when all or the majority of ticks had attached, five

The R. conorii ISF strain was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, U.S.A.).
The rickettsial stock was passaged and maintained in specific
pathogen-free embryonated chicken egg yolk sacs, and passaged three times in Vero cell culture before animal inoculation.
Stocks were prepared from homogenization of infected cells in
sucrosephosphateglutamate buffer (218 mm sucrose, 3.8 mm
KH2 PO4 , 7.2 mm K2 HPO4 , 4.9 mm monosodium glutamic acid,
pH 7.0) and stored at 80 C. The concentration of rickettsiae
in the inoculum was determined by real-time quantitative polymerase chain reaction (qPCR), and diluted in PBS, pH 7.2, prior
to animal inoculation. The mouse inoculum dose, 1 107 copies
of rickettsiae per animal, was based on results from our preliminary infection studies (N. Milhano, unpublished data, 2013) in
C3H/HeJ mice, in which 1 107 copies of rickettsiae per animal
were inoculated i.d., which resulted in consistent infection in the
lungs of all animals.

Quantification of bacterial loads by real-time qPCR

For rickettsial quantification in mouse lung samples and
R. conorii inoculum, DNA was extracted using the Qiagen
DNeasy Blood and Tissue Extraction Kit (Qiagen, Inc., Valencia, CA, U.S.A.). Samples were then analysed by real-time
qPCR targeting Rickettsia spp. citrate synthase gene, gltA, using
primers C5 (forward) and C6 (reverse), as previously described
(Labruna et al., 2004),. Serial dilutions of plasmids containing a single copy of a fragment of R. conorii Malish strain

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Tick saliva in Rickettsia conorii dissemination

gltA gene and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were used for quantification and normalization, respectively. Real-time PCR assays were performed in triplicate using an iCycler IQ from Bio-Rad Laboratories, Inc. (Hercules, CA, U.S.A.) and controls were included in the absence
of DNA template [non-template control (NTC)]. The results are
expressed as rickettsial gltA copies per 106 GAPDH.

Cytokine analysis by real-time reverse transcriptase PCR

Mouse lungs, for both experimental groups and controls,
were collected in RNAlater RNA Stabilization Reagent (Qiagen,
Hilden, Germany) and RNA extraction was performed using the
Qiagen RNeasy Mini Kit (Qiagen, Inc.). cDNA was synthesized
using the Bio-Rad iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc.), and reverse transcriptase (RT)-PCR reactions
were performed in a 25-L final reaction mix, using specific
primers to amplify interleukin-10 (IL-10), IL-1 and NF-B
genes. The -actin gene was used as the housekeeping gene as
a control for variations in RNA concentrations between samples. The RT-PCR assays were performed using an iCycler
IQ (Bio-Rad Laboratories, Inc.) with each reaction including
an NTC. The Ct method was used for quantification and
results expressed as the mRNA relative ratio (2Ct ) (Livak &
Schmittgen, 2001).

Histology
Lung tissue samples from the mice infected with R. conorii,
and either infested or not infested with ticks, were fixed in
10% neutral buffered formaldehyde for a minimum of 24 h and
embedded in paraffin. Tissues were then sectioned at 5 m and
stained with haematoxylin and eosin for histological analysis.

Fig. 1. Real-time quantitative polymerase chain reaction (qPCR) of

rickettsial citrate synthase (gltA) gene. RC/tick infestation, C3H/HeJ
mice infested with ticks and inoculated i.d. with Rickettsia conorii
Israeli spotted fever (ISF) strain; RC, C3H/HeJ mice inoculated i.d. with
R. conorii ISF without tick infestation.

Analysis of cytokine and transcription factor levels in the

lungs revealed that R. conorii inoculation alone induced significantly higher levels of IL-1 and NF-B than those in
R. conorii-infected mice with tick infestation (P < 0.05) (Fig. 2).
By contrast, the tick-infested group had higher IL-10 levels than
the infected mice without tick infestation, but the difference was
not statistically significant (P > 0.05).
Histological sections of the lungs of both groups of
R. conorii-infected animals showed foci of vasculitis with
subendothelial infiltration of inflammatory cells (data not
shown).

Statistical analysis
GraphPad Prism Version 6.0 (GraphPad Software, Inc., La
Jolla, CA, U.S.A.) one-way analysis of variance (anova) and
Students t-test were used to examine statistically significant
differences in rickettsial load, and cytokine and transcription
factor levels between mice infected with R. conorii and infested
with ticks or only inoculated with rickettsiae. P-values of < 0.05
were considered to indicate statistically significant differences in
each analysis.

Results
All mice were killed on day 5 post-inoculation, at which time
the majority of nymphal Rh. sanguineus s.l. had engorged and
detached. At necropsy, visual observation of spleens showed
splenomegaly in R. conorii-infected mice, both with and without
tick infestation. The control mice did not have enlarged spleens.
There was no statistical difference in lung bacterial load between
the two groups of animals (Fig. 1). The controls were negative
for R. conorii infection.

Discussion
In this study, the role of tick saliva in rickettsial transmission in
the host was analysed by i.d. inoculation of R. conorii ISF into
two groups of susceptible animals, one of which was infested
with ticks. There were no statistically significant differences
in bacterial load between the two groups; however, cytokine
and transcriptional analysis did show significant results. The
strain of mice used in this study, C3H/HeJ, had previously
been shown to be susceptible to R. conorii (Eisemann et al.,
1984). This reflects the presence of a missense mutation in the
TLR4 gene of these mice, which leads to a single amino acid
change in the cytoplasmic portion of TLR4. This change results
in a deficiency in TLR4 signalling and a deficient response to
lypopolyssacharide (LPS) (Hoshino et al., 1999). These deficiencies make C3H/HeJ mice highly susceptible to R. conorii
so that they succumb to overwhelming infection at doses that
are non-lethal to TLR4-competent C3H/HeN mice (Jordan et al.,
2009). Because we used this strain of mouse in our study, all
lung samples tested from both experimental groups were found

2015 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12118

4 N. Milhano et al.

(A)

(B)

(C)

Fig. 2. Reverse transcriptase quantitative polymerase chain reaction of mRNA relative ratios (2Ct ) of (A) interleukin-1 (IL-1), (B) IL-10 and
(C) NF-B. RC/tick infestation, C3H/HeJ mice infested with ticks and inoculated i.d. with Rickettsia conorii Israeli spotted fever (ISF) strain; RC,
C3H/HeJ mice inoculated i.d. with R. conorii ISF without tick infestation. *P < 0.05.

to contain rickettsiae; however, there were no statistically significant differences in bacterial load between the groups. The
pathological response was confirmed by histological analysis of
the lungs, in which mild subendothelial cellular infiltration was
observed in the alveolar septa.
Pathogen transmission in the host has been shown to
be enhanced by tick saliva, a phenomenon described as
saliva-activated transmission (Wikel, 1999; Nuttall & Labuda,
2004; Titus et al., 2006). A recent study described experiments
using Rickettsia parkeri and infestations with Amblyomma
maculatum (Ixodida: Ixodidae) ticks to ascertain the role of tick
feeding in rickettsial survival and proliferation at the tick feeding site. This study showed a marked increase in rickettsial load
in the tick-infested group compared with the inoculated-only
group (Grasperge et al., 2012). In this earlier study, mice were
inoculated on the nape of the neck and 20 min later infested with
nymphal ticks at the inoculation site. The present study was
designed to investigate the systemic proliferation of R. conorii.
Therefore, in preliminary experiments (N. Milhano, unpublished data, 2013), we analysed spleen, lung, liver and lymph
nodes for bacterial burdens and detected consistent infection
in the lungs. In the present study, we thus decided to focus on
this organ. Our rationale for allowing the ticks to attach first
and for some salivation to occur into the feeding site and only
then to inoculate the animals with Rickettsia was based on
mimicking what might happen during tick feeding. Once a tick
attaches, saliva is injected into the wound, immediately initiating anti-inflammatory responses which may then allow for the
enhanced transmission of the pathogen (Wikel, 2013). Based on
the established C3H/HeN model for R. conorii, in which peak
rickettsial infections were verified on days 3 and 5 (Walker
et al., 1994), we determined that day 5 would be appropriate for
animal studies after an i.d. inoculation that would be likely to
result in a delay to dissemination compared with i.v. inoculation.
Furthermore, by day 5 most of the nymphal ticks had completed
their bloodmeal and thus the saliva had been allowed to exert its
effects throughout the whole feeding period. There are several
potential explanations for the absence of significant differences

in rickettsial burden between the R. conorii inoculated-only

group and the tick-infested R. conorii inoculated group in
our study, including the possibilities that day 5 is either too
soon for the saliva to exert its effects on the dissemination of
rickettsiae, or too late, and that an equilibrium between the
anti-inflammatory components of the saliva and the immune
responses of the host may already have been reached. A further
experiment to elucidate this point would involve the study of
events at more time-points in order to ascertain whether significant differences might be observed between the experimental
groups.
Interestingly, although there was no indication of a tick saliva
effect on rickettsial burden in the lung, the analysis of mRNA
levels of cytokines and the transcription factor NF-B showed
statistically significant differences in IL-1 and NF-B levels
between infected tick-infested mice and infected-only animals.
Both IL-1 and NF-B were higher in the rickettsiae-inoculated
non-infested mice. Ixodes scapularis (Ixodida: Ixodidae) ticks
have been shown to suppress host macrophage and Th1 lymphocyte cytokine production (Urioste et al., 1994; Wikel &
Bergman, 1997) and to polarize immune responses towards a
Th2 cytokine profile (Ferreira & Silva, 1999; Schoeler et al.,
1999), thereby increasing the likelihood of successful feeding
and pathogen transmission (Wikel & Bergman, 1997). Proinflammatory cytokine production, including of IL-1, depends
upon the NF-B pathway (Li & Verma, 2002). Our results,
which show decreases in both IL-1 and NF-B in tick-infested
infected mice, clearly indicate a role of tick feeding in the inhibition of proinflammatory host responses initiated by a tick-borne
pathogen. Another study also showed that extracts from the
salivary gland of Dermacentor andersoni (Ixodida: Ixodidae)
ticks reduced IL-1 levels during the early phases of tick feeding
(Ramachandra & Wikel, 1992). In addition, we observed higher
mRNA levels of IL-10 in tick-infested mice, although the difference was not statistically significant. This finding has been
observed previously in studies in which concomitant increases
in the Th2 cytokine IL-10 in the spleen of C3H/HeJ mice were

2015 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12118

Tick saliva in Rickettsia conorii dissemination

examined under different conditions of infection (Zeidner et al.,
1997; Ferreira & Silva, 1999; Boppana et al., 2009).
In conclusion, the present study demonstrated that nymphal
Rh. sanguineus s.l. tick saliva suppresses the proinflammatory
response in mice inoculated i.d. with R. conorii ISF.
It would be interesting to extend these studies in other mouse
strains with differing levels of susceptibility to R. conorii, such
as susceptible C3H/HeN, less susceptible Balb/c and highly
resistant C57BL/6 mice, and to include additional time-points
and analyses of other anti-inflammatory and proinflammatory
cytokines.
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Accepted 5 January 2015

2015 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12118