MICROORGANISMS ASSOCIATED WITH HARD TICKS (IXODIDAE):FROM SYMBIONTS TO TICK-BORNE PATHOGENS
Creators: name: PISTONE, DARIO
Publisher: name: Universit� degli Studi di Milano
Year: 2012

description: The research activity I developed during my doctorate can be divided into three parts. First, I investigated the
genetic variability of Ixodes ricinus in Europe and North Africa also screening for the presence of bacteria
belonging to Borrelia burgdorferi sensu latu complex. A second part of the work focused on Midichloria
mitochondrii the principal endosymbiont of I. ricinus. This intramitochondrial bacterium is also present in
other tick species but information on the exact intracellular location are not available for the majority of them.
I decided to perform a molecular screening for detection of Midichloria-like organisms in vertebrates
comprehending humans. Finally, the last part of this work provide information on presence of Ixodes ricinus
and other tick species in areas (and from hosts) where record of the presence of these arthropods were
absent or fragmentary; the ticks collected were also screened for the presence of different tick-borne
pathogens.
The genetic analysis on both two mitochondrial loci (COI and COII) and two nuclear loci (Defensin and
TROSPA) of Ixodes ricinus confirmed the existence of two distinct groups of haplotypes showing a clear
geographic pattern. The first group comprises individuals collected in the European continent, while the
second group comprises individuals collected in Tunisia, North Africa. The existence of two groups of
populations genetically differentiated in the two continents is also supported by the spatial analysis of
molecular variance (SAMOVA), congruently for both mtDNA loci and the two nuclear loci. In previous studies
on European and Northern African populations the lacking of data from intermediate areas did not allow
determining the entity and the nature of the observed discontinuity. In our study, in addition to a considerable
number of individuals from Central and Northern Europe, Southern populations of Ixodes ricinus, sampled in
Italy (Sicily) were included. These individuals belong to European group, hence marking the existence of an
abrupt and strong genetic discontinuity. Several hypotheses had been proposed to explain this pattern. It
could be linked to geographic discontinuity due to the presence of the Mediterranean sea separating the two
continents. Anyway, the absence of discontinuity in the entire European continent and the possibility for I.
ricinus to cover large distance feeding on migratory birds, might make questionable this hypothesis. Besides,
there could be implicated ecological factors linked to I. ricinus biology and interactions with its hosts. Another
hypothesis, not necessary excluding the previous ones, considers the role of interaction between species.
Indeed, I. ricinus is a parasites and a vector of other parasites/pathogens, interacting both with its hosts and
with the transmitted pathogens, and this fact might be the cause of the genetic difference in the two
populations. All these selective pressures might have contributed to the insurgence of the observed
differences between European and Tunisian populations of I. ricinus. Nuclear and mitochondrial loci are
concordant in evidencing a genetic discontinuity between the two continents, but the two markers show
different patterns. For mitochondrial DNA, no Tunisian haplotypes were encountered in Europe and vice
versa. Nuclear loci show a degree of sharing of haplotypes of the two different haplogroups, probably due to
long-distance migration of avian hosts. Further interesting ecological and evolutionary scenarios, in a
speculative way, might be suggested to explain such differences. For example, genetic drift is able to drive
the loss of genetic variants in a totally random process. The flux of haplotypes might involve not only nuclear
genes, but also mitochondrial genes, but these ones are lost due to genetic drift that on this marker is
stronger compared to nuclear loci. An alternative hypothesis proposes the existence of differential migration
of males and females, where males do not transmit the mt genome. Indeed, females of I. ricinus resulted
more philopatric compared to males and less incline to dispersion. Host preference with different dispersion
capacity might explain this pattern, males tend to parasitize more often birds and females are more
frequently encountered on large mammals. Another hypothesis might be linked to a selective disadvantage
of females in the two geographic regions.
In conclusion, the European population of I. ricinus does not present any phileogeographic structure. I would
suggest that the pattern of low genetic diversity observed at nuclear and mitochondrial loci is a consequence
of historical and contemporary factors. Both markers show traces of demographic expansion, in fact,
mismatch distribution resulted unimodal and not deviate from the model expected in case of demographic
expansion. Besides, the result is confirmed from the values of Fu?s Fs index that are negative and significant.
Over crossing the actual distribution of the species I. ricinus and the distribution of ice in glacial phases in
European continent it is reasonable to suppose that the demographic expansion started from glacial refugia.
The value of parameter ? of mismatch distribution of mitochondrial DNA, suggests that this expansion
happened almost 20.000 years ago. Thus, evolutive event might be linked to deep climatic changes in
temperate areas during glacial/interglacial phases in late Pleistocene. During this period, according to the
general model of expansion/contraction, populations of thermophil species, like I. ricinus, to defend from ice
advancing, moved to lower latitudinal refugia in suitable areas for survival, identified in the Southern
Mediterranean peninsulae. In these refugia, various I. ricinus populations survived during glacial ages, and
after ice melting and temperature rising, migrated to Northern areas. Previous studies on I. ricinus,
hypothesized that this model, already validated for other species, is also valid for this parasite. But the
results of this study suggest a different evolutive scenario. In fact, even if the genetic data support the
hypothesis of an event of demographic and spatial expansion after glacial ages, on the contrary, the
absence of different lineages localized inside the European continent, as the absence of a clinal variation of
haplotypes frequencies do not support the glacial genetic fragmentation. The pattern observed is more
adapted to explain a scenario were populations of I. ricinus remained inter-connected probably due to
continuity offered by its multiple hosts, as suggested for other species with wide distribution in Western
Paleartic (or we could even hypothesize that the species survived the glacial age into a single refugium). In
the case of I ricinus is of fundamental importance to consider the ecological characteristics of the species in
order to determine the response in front of the Pleistocenic climatic changes. The present analysis consents
to evidence an historical component at the origin of actual pattern of distribution of genetic diversity, but also
a variety of actual processes that acted on populations. The migration on long distances mediated by
different I. ricinus hosts in different stages of its biological cycle might be implicated in the absence of
correlation between genetic and geographic distances, the presence of haplotypes shared by populations
distantly located and the lack of areas with major and minor diversity. On the contrary, the presence of
specific and private haplotypes in areas in close vicinity suggests a restricted genetic flux on small
geographic scale.
The PCR screening for Borrelia burgdorferi sensu latu on ticks collected in Europe and North Africa allows to
determine the presence of 4 species of borreliae causing human diseases (B. burgdorferi ss, B afzelii,
B.garinii and B. lusitaniae) in the analyzed ticks. Due to the small number of individuals included in this study
it was not possible to derive statistical correlations between the presence of spirochetes and specific
mithocondrial or nuclear gene alleles (COI, COII; Defensin and Trospa). An interesting result of this study
evidence that B. lusitaniae, a genospecies diffused in Portugal, Southern Spain and Northern Africa with a
reduced distribution outside this areas, might be widely diffuse in other European countries (Italy and Czech
Republic) and also in Turkey.
Our analyses confirm that the genetic variability of I. ricinus in Continental Europe and in Northern Africa is
limited. Correspondingly, the genetic variability of its principal endosymbiont Midichloria mitochondrii is also
negligible. We suppose that this microorganism might have established the mutualistic association with I.
ricinus only in recent times and probably experimented a recent increase in population size following the
Paleocenic population expansion of its tick vector host. So far, this idea is not yet supported by proper
investigation. M. mitochondrii was detected in various ticks species that could have acquired the bacteria
during blood meal passing trough the infection of the host or during co-feeding on the vertebrate hosts (i.e.
without the infection of the vertebrate). Besides, M. mitochondrii is present in several tick species apparently
without remarkable variability (i.e., even identical 16s rDNA sequences are observed in different species),
thus suggesting for M. mitochondrii the possibility to reach new hosts/vectors trough horizontal transmission.
The presence of various 16S rDNA gene sequences with high similarity to that of M. mitochondrii in other
Metazoa, including in environmental microbial mats suggests the existence of a family of Midichloria-like
organisms (MLOs). Besides, the detection of DNA of MLOs in other hematophagous arthropods suggest a
possibility of transmission and ?circulation? of these bacteria between vectors and vertebrate hosts. The lack
of congruence between ticks and Midichloria bacteria phylogenies is a further indirect evidence of possible
horizontal transmission. Even in the case of infection of the host, at present, there is no evidence supporting
the pathogenic role of M. mitochondrii in vertebrates, but it is important to remind that several human
mitochondrial dysfunction/pathologies are currently of unknown etiology. Our molecular screening in blood
and tissues of vertebrates (including humans) detected 8 PCR positive blood samples from 4 horses (Equus
caballus), 3 dogs (Canis familiaris) and 1 sheep (Ovis aries), out of a total of 293 mammalian blood samples
examined. The sequences obtained were not identical; they showed high similarity with the 16S rDNA of
MLOs detected in species different from I. ricinus.
In these three years, my doctorate work on tick-borne pathogens (TBPs) contributed in providing new
information on the presence of ticks and associated pathogenic microorganisms in Italian areas were
knowledge were scanty, incomplete or obtained indirectly from hospitalized patients presenting clinical
manifestations due to a specific TBD.
In the framework of the investigation on tick-borne pathogens, I reported the presence of spirochetes of the
species Borrelia afzelii, implicated in development of Lyme disease in humans, and B. lusitaniae, also
suspected to be pathogenic for humans, in an highly populated area close to the industrial district of Milan
(Ticino Park and surrounding area). This investigation allowed to describe human cases of borreliosis in a
zone previously not consider at risk of tick infestation or endemic for Lyme disease. We also contributed in
the process of valuation of the risk and management of the public health problem, providing correct
information to clinicians, workers/tourists of the area exposed to the risk of tick-bite and defining the ?red?
zones/hot spots at high risk where it is necessary to position informative signs on tick presence and tickborne
diseases transmitted. Further study in the Ticino Park area detected the presence of Rickettsia
monacensis and R. helvetica (both belonging to Spotted Fever Group-SFG rickettsiae) with high prevalence
in Ixodes ricinus; and also the circulation of Francisella tularensis holartica (Type B) was reported. After this
study, SFG rickettsi.