Humans parasitized by the hard tick Ixodes ricinus are seropositive to Midichloria mitochondrii: Is Midichloria a novel pathogen, or just a marker of tick bite?

Article (PDF Available) · November 2012with37 Reads
DOI: 10.1179/2047773212Y.0000000050 · Source: PubMed
Abstract
Midichloria mitochondrii is an intracellular bacterium found in the hard tick Ixodes ricinus. In this arthropod, M. mitochondrii is observed in the oocytes and in other cells of the ovary, where the symbiont is present in the cell cytoplasm and inside the mitochondria. No studies have so far investigated whether M. mitochondrii is present in the salivary glands of the tick and whether it is transmitted to vertebrates during the tick blood meal. To address the above issues, we developed a recombinant antigen of M. mitochondrii (to screen human sera) and antibodies against this antigen (for the staining of the symbiont). Using these reagents we show that (i) M. mitochondrii is present in the salivary glands of I. ricinus and that (ii) seropositivity against M. mitochondrii is highly prevalent in humans parasitized by I. ricinus (58%), while it is very low in healthy individuals (1·2%). These results provide evidence that M. mitochondrii is released with the tick saliva and raise the possibility that M. mitochondrii is infectious to vertebrates. Besides this, our study indicates that M. mitochondrii should be regarded as a package of antigens inoculated into the human host during the tick bite. This implies that the immunology of the response toward the saliva of I. ricinus is to be reconsidered on the basis of potential effects of M. mitochondrii and poses the basis for the development of novel markers for investigating the exposure of humans and animals to this tick species.
Humans parasitized by the hard tick Ixodes
ricinus are seropositive to Midichloria
mitochondrii:isMidichloria a novel pathogen,
or just a marker of tick bite?
Mara Mariconti
1,2
, Sara Epis
1
, Paolo Gaibani
3
, Claudia Dalla Valle
2
,
Davide Sassera
1
, Paola Tomao
4
, Massimo Fabbi
5
, Francesco Castelli
6
,
Piero Marone
2
, Vittorio Sambri
3
, Chiara Bazzocchi
1
, Claudio Bandi
1
1
Dipartimento di Scienze Veterinarie e Sanita
`
Pubblica, Universita
`
degli Studi di Milano, Milano, Italy,
2
Fondazione IRCCS Policlinico San Matteo, Pavia, Italy,
3
U.O. Microbiologia CRREM, Azienda Ospedaliera
Universitaria S.Orsola-Malpighi, Bologna, Italy,
4
INAIL Department of Occupational Medicine, Roma, Italy,
5
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Sezione Diagnostica di Pavia,
Italy,
6
Clinica di Malattie Infettive e Tropicali, Universita
`
degli Studi di Brescia, Brescia, Italy
Midichloria mitochondrii is an intracellular bacterium found in the hard tick Ixodes ricinus. In this arthropod,
M. mitochondrii is observed in the oocytes and in other cells of the ovary, where the symbiont is present in
the cell cytoplasm and inside the mitochondria. No studies have so far investigated whether M.
mitochondrii is present in the salivary glands of the tick and whether it is transmitted to vertebrates during
the tick blood meal. To address the above issues, we developed a recombinant antigen of M. mitochondrii
(to screen human sera) and antibodies against this antigen (for the staining of the symbiont). Using these
reagents we show that (i) M. mitochondrii is present in the salivary glands of I. ricinus and that (ii)
seropositivity against M. mitochondrii is highly prevalent in humans parasitized by I. ricinus (58%), while it is
very low in healthy individuals (1.2%). These results provide evidence that M. mitochondrii is released with
the tic k saliva and raise the possibility that M. mitochondrii is infectious to vertebrates. Besides this, our
study indicates that M. mitochondrii should be regarded as a package of antigen s inoculated into the
human host during the tick bite. This implies that the immunology of the response toward the saliva of I.
ricinus is to be recon sidered on the basis of potential effects of M. mitochondrii and poses the basis for the
development of nov el markers for investigating the exposure of humans and animals to this tick species.
Keywords: Ixodes ricinus, Midichloria mitochondrii, Human sera, Tick-borne bacteria, MALOs, Serological screening
Introduction
Midichloria mitochondrii is an intracellular bacterium
found in the hard tick Ixodes ricinus.
1
In this
arthropod, M. mitochondrii is abundant in diverse
cell types of the ovary, including oocytes.
2
No
evidence has so far been published on the presence
of this bacterium in the salivary glands of ticks. M.
mitochondrii is peculiar in that it is observed not only
in the cell cytoplasm, but also inside the mitochon-
dria, within the intermembrane space of these
organelles.
1,2
In I. ricinus, M. mitochondrii is verti-
cally transmitted to the progeny, as indicated by PCR
evidence on eggs and newly-emerged larvae and by
the presence of these bacteria in the oocytes of this
tick.
1,2
Bacteria closely related to M. mitochondrii
have also been detected in other tick species;
3,4
in
addition, 16S rRNA g ene sequences that cluster with
M. mitochondrii have been amplified from a variety
of sources, including fleas, bed bugs, tabanid flies
and cnidarians.
5–9
Since M. mitochondrii is the first
organism formally described for this bacterial
cluster,
1
and since taxonomic revision for this cluster
has not yet been published, we will refer to this group
of bacteria as Midichloria and like organisms (here-
after: MALOs). There is circumstantial evidence that
MALOs could be transmitted to terrestrial verte-
brates during the tick bite:
(i) 16S rRNA gene sequences related with M.
mitochondrii have been amplified from roe deer
during a screening for tick-borne bacteria;
10
(ii) phylogeny of ticks and their respective MALOs
are not congruent, with distantly related ticks
Correspondence to: C. Bandi, Dipartimento di Scienze Veterinarie e
Sanita` Pubblica, Universita` degli Studi di Milano, Milano, Italy. Email:
claudio.bandi@unimi.it
ß
W. S. Maney & Son Ltd 2012
MORE Ope nChoice articles are ope n access and di stributed under the terms of the Creative Commons Attr ibution License 3.0
DOI 10.1179/2047773212Y.0000000050
Pathogens and Global Heal th 2012 VOL.106 NO.7 391
harboring MALOs that are identical at the 16S
rRNA gene level;
3
this implies that MALOs
could be transmitted horizontally among ticks,
and a simple mechanism that could be hypothe-
sized to explain horizontal transmission is the
infection of an host parasitized by different tick
species (or co-feeding with bacterial transmission
without true infection);
(iii) MALO 16S rRNA gene sequences have been
amplified from human patients parasitized by ticks.
11
Based on the above information we designed a study
to investigate whether M. mitochondrii is present in
the salivary glands of the host tick I. ricinus and
whether humans parasitized by I. ricinus develop
antibodies against M. mitochondrii. To address the
above questions, we used a recombinant antigen from
M. mitochondrii and a polyclonal antibody raised
against this antigen (described in Mariconti et al.).
12
We first used the antibody for immunostaining on
tick salivary glands and then we used the recombi-
nant antigen for the analysis of human sera, from
healthy blood donors and from persons parasit-
ized by ticks. We also used PCR for searching of
M. mitochondrii DNA in tick sali vary glands and
rostra.
Materials and Methods
Tick samples
Three semi-engorged adult females of I. ricinus and
two of Ixodes hexagonus were collected from natu-
rally infected animals (sheep and hedgehog), in the
counties of Varese and Bergamo (Northern Italy).
Ticks were identified morphologically with standard
taxonomic keys.
13
Salivary glands and rostra were
dissected in sterile conditions and prepared for
indirect immunofluorescence assay (salivary glands)
or for PCR analysis (salivary glands and rostra).
DNA extraction and PCR analysis
DNA from salivary glands and rostra was extracted
using the DNeasy Blood & Tissue Kit (Qiagen,
Hilden, Germany), eluted in 50 ml of sterile water,
quantified by a spectrophotometer and stored at
280uC before use. PCR was affected using a 16S
rDNA-targeted semi-nested procedure.
3
Amplified
bands were sequenced using ABI technology, to
confirm the specificity of the amplification.
Sera samples
A total of 249 samples of human sera were screened
for the presence of anti-M. mitochondrii antibodies,
using an expe rimental ELISA assay (see below).
These sera were from two groups of subjects: 169
healthy blood donors and 80 subjects exposed to tick
bite. In particular, the latter group was composed of
persons parasitized by ticks, examined at the emer-
gency services of the hospitals IRCCS Policlinico San
Matteo (Pavia), Azienda Ospedaliera Universitaria
S.Orsola-Malpighi (Bologna) and Spedali Civili di
Brescia (Brescia), or with a reliable request for Lyme
disease diagnosis, based on clinical signs and
anamnesis (of these, 31 had actually been shown to
be seropositive for Borrelia burgdorferi sensu lato
before the enrollment in this study; in the context of
this study, a furt her subject was then shown to
be seropositive for B. burgdorferi see below).
Unfortunately, in most cases the emergency service
physicians did not conserve the ticks removed from
the patients for identification; when the tick was
identified, it was in most cases I. ricinus (13 out of 15
ticks examined). Based on this information, on a
previous study showing that 90% of the ticks
removed from humans in Northern Italy are I.
ricinus,
14
and on preliminary results of a parallel
study in the same area (Bandi et al., unpublished
results), we assume that most of the patients had been
parasitized by I. ricinus. All of the above sera were
collected after no less than six weeks from the
removal of the tick. The screening on human sera
was conducted under the regulation of the Ethical
Committees of the S. Orsola Malpighi University
Hospital (Bologna), Spedali Civili di Brescia and
Fondazione IRCCS Policlinico San Matteo (Pavia);
all patients provided informed consent and the study
protocol was approved by the Ethical Committees of
the above hospitals.
Indirect immunofluorescence assay on tick
salivary glands
Salivary glands were stained with MitoTracker Red
CMXRos (Invitrogen Carlsbad, CA, USA) and with a
polyclonal antibody raised against the recombinant
flagellar FliD protein of M. mitochondrii (anti-rFliD)
as previously described.
12
Observation was recorded
with a Leica confocal microscope (LeicaTCSNT,
Solms, Germany).
Detection of anti-M. mitochondrii antibodies in
human sera
The recombinant flagellar protein FliD of M.
mitochondrii (rFliD) was produced in Escherichia coli
and purified as previously described.
12
Anti-rFliD
antibody levels in human sera were determined using
an enzyme-linked immunosorbent assay (ELISA),
using 96-well microtiter plates coated with 0.1 mg/well
of rFliD protein. Each sample was diluted 1/100 in
phosphate buffered saline supplemented with 1%
bovine serum albumin and 100 ml of each diluted sera
were tested following the procedure previously
described in Gaibani et al.
15
Threshold value was
established as the mean optical density (OD)
450/630
of
the sera from the healthy blood donor s plus three
times the standard deviation (i.e. mean OD
450/630
z3
standard deviations). Using this method the thresh-
old was set at 0.793. Each sample was considered
negative if its OD
450/630
was less than the threshold
Mariconti et al. Is Midichloria a novel pathogen?
392 Pathogens and Global Health 2012 VOL.106 NO.7
value, and positive if its OD
450/630
was higher than or
equal to the threshold.
Serological screening for B. burgdorferi
Even though part of the sera used in this study had
already been diagnosed for B. burgdorferi infection, we
examined all of the sera for the presence of IgG anti-
bodies specific for B. burgdorferi. This screening was
performed using a commercial Western blot kit
(Borrelia ViraStripe Test Kit IgG; Viramed Biotech,
Planegg, Germany). Western blot results were inter-
preted following the manufacturer’s recommendations.
Experimental Western blot assays for B.
burgdorferi and M. mitochondrii
AcultureofB. burgdorferi was pelleted with
centrifugation at 4000g for 5 minutes, and the pellet
was suspended in 150 ml of Tris HCl 25 mM (pH 8);
after sonication to promote the release of the
proteins, the supernatant was recovered after cen-
trifugation at 16 000g for 10 minutes at 4uC. The
soluble proteins in the supernatant of B. burgdorferi
or the purified rFliD were then used as antigens for
Western blot assays, performed according to stan-
dard procedures,
16
with dilution of secondary anti-
human antibodies at 1 : 5000. To test the possible
cross-reactivity of rFliD in patients infected by B.
burgdorferi s.l., the following sera were examined, at
1:1000 dilution: five sera positive for B. burgdorferi;
five sera positive for M. mitochondrii; five sera
positive for both B. burgdorferi and M. mitochondrii.
Serological positivity to M. mitochondrii and B.
burgdorferi were determined using respectively the
ELISA method and the Western blot kit described in
the above paragraphs.
Results and Discussion
The indirect immunofluorescence assay on salivary
glands of adult I. ricinus females using a primary
antibody directed against the rFliD of M. mitochon-
drii revealed the presence of intensely green-stained
bodies (Fig. 1ad); these were generally collected in
clusters, and closely associated with mitochondria (as
revealed by the staining using MitoTracker Red). No
staining was observed using the anti-rFliD antibody
on the salivary glands of I. hexagonus (Figure 1eh),
a tick closely related to I. ricinus that does not harbor
M. mitochondrii. No stained bodies were observed in
I. ricinus when the primary anti-rFliD antibody was
not used (not shown). The above observations were
obtained on all the samples examined for I. ricinus
(three positive, out of three adult females) and for I.
hexagonus (two negative, out of two adult females).
Based on the above observations we assume that the
bodies recognized by the anti-rFliD antibody in the
salivary glands of I. ricinus are M. mitochondrii
bacteria, or aggregates of the FliD protein from this
bacterium. PCR analysis with primers specific for M.
mitochondrii was also congruent with the above
results: amplification of M. mitochondrii DNA was
obtained from all three salivary gland samples from I.
ricinus. In addition, PCR on the rostra was positive
for M. mitochondrii in two of the three adult females
of I. ricinus examined (all of the PCR pro ducts
obtained were sequenced, and matched the 16S
rRNA sequence of M. mitochondrii). PCRs on the
Figure 1 Indirect immunofluorescence assay (FITC-conjugated secondary antibodies) on salivary glands from Ixodes ricinus
(ad) and Ixodes hexagonus (eh) semi-engorged adult females; (a, e) staining using polyclonal antibodies raised against the
rFliD from Midichloria mitochondrii (green); (b, f) live mitochondria stained with MitoTracker Red CMXROS (red); (c, g) cellular
nuclei stained for cell viability with TOTO-3 iodide; (d, h) merging of the images. In d the yellow spots indicate the overlap
between M. mitochondrii (green) and mitochondria (red). Colour version available online.
Mariconti et al. Is Midichloria a novel pathogen?
Pathogens and Global Heal th 2012
VOL.106 NO.7 393
salivary glands and rostra were negative on all of the
samples from I. hexagonus. Taken together, the
results of imm unostaining and PCR on the salivary
glands show that M. mitochondrii (or proteins and
DNA from M. mitocho ndrii) is present in the salivary
glands of I. ricinus; PCR positivity on the rostra
indicates that this bacterium (or DNA from this
bacterium) could be released with the saliva.
The above results prompted us to investigate
whether humans parasitized by I. ricinus are seropo-
sitive for M. mitochondrii. To this purpose, we used
the flagellar rFliD protein from M. mitochondrii as an
antigen, in an ELISA screening on healthy blood
donors and on subjects exposed to tick bite (Fig. 2).
In tick-exposed subjects, the average OD values
for IgG antibodies reacting with rFliD was 0.845
(SD50.422); in healthy blood donors, OD values
were significantly lower (U-MannWitney test;
P,0.001), with an average of 0.373 (SD50.140).
After setting a threshold at an OD value of 0.793 (see
Methods), we could then estimate that the seropre-
valence for M. mitochondrii was 58.75% in subjects
exposed to tick bite (47 out of 80), and 1.18% in the
healthy blood donors (2 out of 169). These prevalence
values are significantly different between the two
groups (U-MannWitney test; P,0.001). The above
results clearly ind icate that subjects exposed to tick
bite produce antibodies that react with an antigen
from M. mitochondrii, and indicate that this bacter-
ium is inoculated into the human host during the tick
bite.
The antigen that we used for the above ELISA
screening (rFliD) corresponds to a portion of a
flagellar protein of M. mitochondrii. Thus, we did
not expect any cross-reactivity towards antibodies
generated during an infection caused by any other
Rickettsiales. Indeed, M. mitochondrii is the sole
member of the order Rickettsiales that has so far been
shown to possess flagellar genes. However, I. ricinus
is the main vector in Europe of the Lyme disease
spirochetes (B. burgdorferi s.l.),
17
bacteria that possess
flagella, well known for their immunogenicity.
18
One
could thus argu e that production of IgG antibodies in
tick-exposed subjects had been induced by a Borrelia
infection, and that these antibodies cross-reacted with
rFliD from M. mitochondrii. Considering the numer-
ous amino-acid differences between the FliD proteins
of the two bacteria, we consider cross-reactivity
rather unlikely.
12
However, we de cided to address
this issue by screening all of the sera included in this
study with a Western blot diagnostic kit for the
detection of antibodies against B. burgdorferi s.l.
Using this kit, positivity for B. burgdorferi s.l. was
revealed in 32 out of the 80 samples of sera from the
subjects exposed to tick bite; none of the healthy
blood donors was positive to B. burgdorferi s.l.
Figure 2 Results of the serological screening for Midichloria mitochondrii and Borrelia burgdorferi on sera from 80 tick-
exposed subjects (main figure) and from 169 healthy blood donors (inset). M: sera positive to M. mitochondrii; B: sera positive
to B. burgdorferi;M
z
B: sera positive to both M. mitochondrii and B. burgdorferi. Sera negative to both bacteria (NEGATIVE)
are also indicated.
Mariconti et al. Is Midichloria a novel pathogen?
394 Pathogens and Global Health 2012 VOL.106 NO.7
Among the 32 subjects positive to B. burgdorferi s.l.,
11 were concurrently positive to M. mitochondrii;
however, a total of 36 subjects positive to M.
mitochondrii were negative to B. burgdorferi.
Finally, 21 subjects were positive only to B.
burgdorferi (Fig. 2). In summary, the above results
show that a high proportion of the subjects positive
to M. mitochondrii were not positive to B. burgdorferi
(and vice-versa), indicating that the positivity to the
former bacterium does not derive from the cross-
reactivity with the latter.
We further addressed the above issue using an
experimental Western blot assay, using as antigens
rFliD from M. mitochondrii and proteins from B.
burgdorferi s.l. This Western blot assay was carried
out on five sera each from the following types of
subjects: positive for M. mitochondrii, positive for B.
burgdorferi s.l., and positive for both bacteria. The
following results were obtained. Sera from the first
five patient labeled one band at 38 kDa, correspond-
ing to the molecular weight of rFliD; no band
corresponding to the molecular weight of B. burg-
dorferi FliD (78 kDa) was observed in these subjects.
The sera from the patients positive to B. burgdorferi
s.l. reacted with a band at 78 kDa (corresponding to
B. burgdorferi FliD), while no labeling was observed
at 38 kDa. Sera from patients that were positive both
for B. burgdorferi s.l. and M. mitochondrii labeled
bands at both 78 and 38 kDa (results not shown).
These results indicate that the antibodies raised
against the FliD protein of B. burgdorferi did not
react with the homologous protein of M. mitochon-
drii, and vice-versa .
Conclusions
Our work provides strong evidence for the transmis-
sion of M. mitochondrii to humans during the blood
meal of I. ricinus. Based on the results here reported,
we cannot conclude that M. mitochondrii replicates
in the human host, determining a true infection.
However, should we assume that M. mitochondrii
does not replicate into the human host, we would
have to conclude that the amount of bacteria (or
bacterial antigens) inoculated is by itself sufficient for
stimulating an antibody production. Overall, we are
more prone to hypothesize that live M. mitochondrii
bacteria (and not just proteins/DNA) can be inocu-
lated into the vertebrate host, and that some
replication can occur therein. At any case, our work
shows that M. mitochondrii is to be regarded not only
as an important symbiont of I. ricinus, but also as a
package of antigens that ticks can inoculate into
vertebrate hosts, and as a potential tick-borne
microorganism that deserves further investigations.
Among the 80 tick-exposed patients that we exam-
ined in this study, 47 were seropositive to M.
mitochondrii, according to the defined threshold
value. We emphasize that we cannot be certain that
all of the subjects had been parasitized by I. ricinus;in
addition, for some of the subjects, the duration of
the blood meal could have been insufficient for an
effective inoculation of bacteria. Furthermore, some
I. ricinus nymphs present a very low M. mitochondrii
load,
19
thus possibly resulting in inoculation of a low
amount of bacteria during their blood meal. These
considerations could explain why not all of the
parasitized subjects were seropositive for this bacter-
ium. On the other hand, immunostaining and PCR
for M. mitochondrii on the salivary glands of I. ricinus
were positive in all three specimens examined.
Since we still do not know whether M. mitochondrii
replicates into the human host, it would be premature
to discuss whether this bacterium could be respon-
sible for any pathological alteration. For sure,
considering the high seroprevalence for M. mitochon-
drii that we determined in tick-exposed subjects, we
would conclude that this bacterium does not cause
overt pathology in humans, at least in the vast
majority of the cases. On the other hand, the high
seroprevalence that we recorded in tick-exposed
subjects (associated with the extremely low seropre-
valence in healthy blood donors) raises the possibility
that M. mitochondrii plays a role in the immune
response and immune-modulation determined by the
I. ricinus saliva, which is important both for the
success of the tick blood meal and for the establish-
ment of the infection by the pathogens vectored by
the tick.
20,21
We emphasize that in the case of filarial
nematodes, the discovery of Wolbachia bacterial
endosymbionts in these parasites and of their
immunological role led to a profound re-thinking of
the immunology of filarial diseas es.
22–24
Finally, anti-
Midichloria antibodies can now be considered as
potential serological markers for I. ricinus bite. Such
markers could be extremely useful to determine the
risk of infection by I. ricinus-borne pathogens in
given areas, and for investigating the epidemiological
association of a variety of pathological alterations
with parasitism by this tick. The present study was
focused on M . mitochondrii, but other MALOs could
possibly be transmitted by ticks and other arthropods
to a variety of vertebrates, including humans (see
introduction). Further studies are now urgent, to
determine whether MALOs represent a novel class of
emerging infectious agents.
Acknowledgements
We thank Micaela Brandolini, Massimo Pajoro,
Dario Pistone and Lina Tomasoni for their help with
the collection of sera and ticks. This work was
partially supported by a grant by Fondazione IRCCS
Policlinico San Matteo, 2012 to PM, and by MIUR
Mariconti et al. Is Midichloria a novel pathogen?
Pathogens and Global Heal th 2012
VOL.106 NO.7 395
Prin (to ChB). Confocal observations have been
carried out thanks to the facilities of CIMA,
Universita` degli Studi di Milano.
References
1 Sassera D, Beninati T, Bandi C, Bouman EA, Sacchi L, Fabbi
M, et al.‘Candidatus Midichloria mitochondrii’, an endosym-
biont of the tick Ixodes ricinus with a unique intramitochondrial
lifestyle. Int J Syst Evol Microbiol. 2006;56:2535–40.
2 Sacchi L, Bigliardi E, Corona S, Beninati T, Lo N, Franceschi
A. A symbiont of the tick Ixodes ricinus invades and consumes
mitochondria in a mode similar to that of the parasitic
bacterium Bdellovibrio bacteriovorus. Tissue Cell. 2004;36:43–
53.
3 Epis S, Sassera D, Beninati T, Lo N, Beati L, Piesman J, et al.
Midichloria mitochondrii is widespread in hard ticks (Ixodidae)
and resides in the mitochondria of phylogenetically diverse
species. Parasitology. 2008;135:485–94.
4 Beninati T, Riegler M, Vilcins IM, Sacchi L, McFadyen R,
Krockenberger M, et al. Absence of the symbiont Candidatus
Midichloria mitochondrii in the mitochondria of the tick Ixodes
holocyclus. FEMS Microbiol Lett. 2009;299:241–7.
5 Erickson DL, Anderson NE, Cromar LM, Jolley A. Bacterial
communities associated with flea vectors of plague. J Med
Entomol. 2009;46:1532–6.
6 Richard S, Seng P, Parola P, Raoult D, Davoust B, Brouqui
P. Detection of a new bacterium relat ed to Candidatus
Midichloria mitochondrii in bed bugs. Clin Microbiol Infect.
2009;15:84–5.
7 Hornok S, Fo¨ldva´ ri G, Elek V, Naranjo V, Farkas R, de la
Fuente J. Molecular identification of Anaplasma marginale and
ric kettsial endosymbionts in blood-sucking flies (Dipt era:
Tabanidae, Muscidae) and hard ticks (Acari: Ixodidae). Vet
Parasitol. 2008;154:354–9.
8 Matsuura Y, Kikuchi Y, Meng XY, Koga R, Fukatsu T. Novel
clade of alphaproteobacterial endosymbionts associated with
stinkbugs and other arthropods. Appl Environ Microbiol.
2012;78:4149–56.
9 Sunagawa S, Woodley CM, Medina M. Threatened corals
provide underexplored microbial habitats. PLoS One. 2010;
5:e9554.
10 Ska rphe´dinsson S, Jensen PM, Kristiansen K. Survey of
tickborne infections in Denmark. Emerg Infect Dis. 2005;
11:1055–61.
11 Mediannikov OIu, Ivanov LI, Nishikawa M, Saito R,
Sidel’nikov IuN, Zdanovskaia NI, et al. Microorgani sm
‘Montezuma’ of the order Rickettsiales: the potential causative
agent of tick-borne disease in the Far East of Russia. Zh
Mikrobiol Epidemiol Immunobiol. 2004;1:7–13.
12 Mariconti M, Epis S, Sacchi L, Biggiogera M, Sassera D,
Genchi M, et al. A study on the presence of flagella in the order
Rickettsiales: the case of Candidatus Midichloria mitochondrii’.
Microbiology. 2012;158:1677–83.
13 Manilla G (editor). Acari Ixodida. Bologna: Ed Calderini; 1998.
14 Manfredi MT, Dini V, Piacenza S, Genchi C. Tick species
parasitizing people in an area endemic for tick-borne diseases in
north-western Italy. Parassitologia. 1999;41:555–60.
15 Gaibani P, Pierro A, Alicino R, Rossini G, Cavrini F, Landini
MP, et al. Detection of Usutu-virus-specific IgG in blood
donors from northern Italy. Vector Borne Zoonotic Dis.
2012;12:431–3.
16 Sambri V, Marangoni A, Eyer C, Reichhuber C, Soutschek E,
Negosanti M, et al. Wes tern immunoblotting with five
Treponema pallidum recombinant antigens for serologic diag-
nosis of syphilis. Clin Diagn Lab Immunol. 2001;8:534–9.
17 Stanek G, Strle F. Lyme borreliosis. Lancet. 2003;15:1639–47.
18 Aguero-Rosenfeld ME, Wang G, Schwartz I, Wormser GP.
Diagnosis of lyme borreliosis. Clin Microbiol Rev. 2005;
18:484–509.
19 Sassera D, Lo N, Bouman EA, Epis S, Mortarino M, Bandi C.
Candidatus Midichloria endosymbionts bloom after the blood
meal of the host, the hard tick Ixodes ricinus. Appl Environ
Microbiol. 2008;74:6138–40.
20 Fontaine A, Diouf I, Bakkali N, Misse´ D, Page`s F, Fusai T,
et al
. Implication of haematophagous arthropod salivary
proteins in host-vector interactions. Parasit Vectors. 2011;
28:187.
21 Lieskovska J, Kopecky J. Effect of tick saliva on signalling
pathways activated by TLR-2 ligand and Borrelia afzelii in
dendritic cells. Parasite Immunol. 2012;34:421–9.
22 Bandi C, Anderson TJ, Genchi C, Blaxter ML. Phylogeny of
Wolbachia in filarial nematodes. Proc Biol Sci. 1998;265:2407–
13.
23 Bazzocchi C, Ceciliani F, McCall JW, Ricci I, Genchi C, Bandi
C. Antigenic role of the endosymbionts of filarial nematodes:
IgG response against the Wolbachia surface protein in cats
infected with Dirofilaria immitis. Proc Biol Sci. 2000;267:2511–
6.
24 Taylor MJ, Bandi C, Hoerauf A. Wolbachia bacterial endo-
symbionts of filarial nematodes. Adv Parasitol. 2005;60:245–84.
Mariconti et al. Is Midichloria a novel pathogen?
396 Pathogens and Global Health 2012 VOL.106 NO.7
    • "In summary, our current work on animals, together with the previously published study on humans [11], presents overall evidence that ticks inoculate Midichloria bacteria into mammalian hosts during the blood meal, and that these bacteria likely multiply, inducing an antibody response . Whether inoculation of Midichloria bacteria could lead to the infection of the host, and whether this could determine pathological alterations, is now a main research issue. "
    [Show abstract] [Hide abstract] ABSTRACT: The Midichloriaceae is a novel family of the order Rickettsiales, that encompasses intracellular bacteria associated with hard ticks (Ixodidae) and other arthropods. The most intensively investigated member of this family is Midichloria mitochondrii, a symbiotic bacterium of the sheep tick Ixodes ricinus, characterized by the capacity of multiplying inside the mitochondria. A recent study suggested that these bacteria might be inoculated into the human host during the tick bite. The purpose of this study was to determine the potential infectivity of Midichloria bacteria for non-human animals exposed to the risk of tick bite. Blood from horses, cattle, sheep and dogs exposed to the risk of tick bite was included in this study. DNAs were extracted, and amplified using 16S ribosomal RNA primers conserved in the Midichloria genus. Furthermore, sera from dogs exposed to the risk of tick bite were analyzed in order to evaluate the presence of antibodies against the recombinant flagellar protein (rFliD) from M. mitochondrii using an ELISA test. Here we present two lines of evidence that support the possibility that bacteria from the genus Midichloria are inoculated into vertebrate hosts during a tick bite: (i) a direct evidence, i.e. the detection of circulating DNA from bacteria related with M. mitochondrii, in the blood of vertebrates exposed to tick parasitism; (ii) a further indirect evidence, i.e. the presence of antibodies against an antigen from M. mitochondrii in dogs exposed to the risk of tick bite. It is interesting to note that variability was detected in the Midichloria gene sequences recovered from positive animals, and that some of these sequences were identical to those generated from tick-associated Midichloria. Based on the results, and on the overall information so far published on the genus Midichloria, we suggest that these bacteria are likely to represent a novel group of vector-borne agents, with the potential of infecting mammalian hosts. Whether inoculation of Midichloria bacteria could cause a true infection and pathological alteration in mammalian hosts is still to be determined. Surely, results emphasize the relevance of Midichloria bacteria in investigations on tick immunology and tick-bite markers.
    Full-text · Article · Dec 2013
    • "These endosymbiotic bacteria persistently infect all life cycle stages of the ticks and are passed on to the next generation transovarially. It is unclear whether or not bacterial endosymbionts are transmitted to vertebrates during tick feeding; a recent study of humans bitten by Ixodes ticks suggests that salivary transmission of the intramitochondrial tick symbiont Candidatus Midichloria mitochondrii (Sassera et al., 2006) can occur to a level sufficient to induce production of specific antibodies (Mariconti et al., 2012). In contrast, Rickettsia peacockii, an endosymbiont of the tick Dermacentor andersoni, was not pathogenic for laboratory animals and failed to infect a range of mammalian cells in vitro (Kurtti et al., 2005). "
    [Show abstract] [Hide abstract] ABSTRACT: Ticks transmit a wide range of viral, bacterial and protozoan pathogens, many of which can establish persistent infections of lifelong duration in the vector tick and in some cases are transmitted transovarially to the next generation. In addition many ixodid and argasid tick cell lines and, by inference the parent ticks from which they were derived, harbor endogenous viruses (ETV) of which almost nothing is known. In general, low level persistent infections with viral pathogens (arboviruses) are not known to have a deleterious effect on tick survival and fitness, suggesting that they can strike a balance with the tick innate immune response. This tolerance of arbovirus infection may be modulated by the permanent presence of ETV in the host cell. In mosquito cells, temporary or permanent silencing of the genes of an endogenous virus by RNA interference can result in changes in replication rate of a co-infecting arbovirus. We propose that tick cell lines offer a useful model system for in vitro investigation of the modulatory effect of ETV on superinfecting pathogen survival and replication in ticks, using the molecular manipulation techniques applied to insect cells.
    Full-text · Article · Jul 2013
  • [Show abstract] [Hide abstract] ABSTRACT: “Candidatus Midichloria mitochondrii” is an intramitochondrial bacterium of the order Rickettsiales associated with the sheep tick Ixodes ricinus. Bacteria phylogenetically related to “Ca. Midichloria mitochondrii” (midichloria and like organisms [MALOs]) have been shown to be associated with a wide range of hosts, from amoebae to a variety of animals, including humans. Despite numerous studies focused on specific members of the MALO group, no comprehensive phylogenetic and statistical analyses have so far been performed on the group as a whole. Here, we present a multidisciplinary investigation based on 16S rRNA gene sequences using both phylogenetic and statistical methods, thereby analyzing MALOs in the overall framework of the Rickettsiales. This study revealed that (i) MALOs form a monophyletic group; (ii) the MALO group is structured into distinct subgroups, verifying current genera as significant evolutionary units and identifying several subclades that could represent novel genera; (iii) the MALO group ranks at the level of described Rickettsiales families, leading to the proposal of the novel family “Candidatus Midichloriaceae.” In addition, based on the phylogenetic trees generated, we present an evolutionary scenario to interpret the distribution and life history transitions of these microorganisms associated with highly divergent eukaryotic hosts: we suggest that aquatic/environmental protista have acted as evolutionary reservoirs for members of this novel family, from which one or more lineages with the capacity of infecting metazoa have evolved.
    Full-text · Article · Mar 2013
Show more