Tiziana Beninati

A Novel Alpha-Proteobacterium Resides in the Mitochondria of Ovarian Cells of the Tick Ixodes ricinus

ABSTRACT An intracellular bacterium from Ixodes ricinus ticks collected in Italy was characterized by electron microscopy (EM), PCR sequencing of the 16S rRNA gene, molecular phylogenetic analysis, and in situ hybridization (ISH). This bacterium was shown by EM to be present in the cytoplasm, as well as in the mitochondria of ovarian cells. When universal 16S rRNA bacterial primers were used, PCR amplification of ovarian DNA followed by cloning and sequencing resulted in the same sequence being found in each sample. Phylogenetic analysis of this sequence showed that the bacterium from which it was derived, tentatively designated IricES1, is part of a novel clade in the alpha subdivision of the Proteobacteria. ISH and PCR assays of various tissues performed with oligonucleotides specific for the IricES1 16S rRNA showed that IricES1 is restricted to ovarian cells. Based on the results obtained, we inferred that the bacteria seen by EM in ovarian cells are a single type of bacteria, corresponding to IricES1. PCR screening of 166 ticks from various parts of Italy and one site in England showed that IricES1 was present in 96% of adult females and 44% of nymphs (unsexed). No adult males were found to be infected. Despite the apparent parasitism of host mitochondria by IricES1, the available information suggests that the bacterium has an obligate relationship with its host, although this must be confirmed.

Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species

The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.

First detection of spotted fever group rickettsiae in Ixodes ricinus from Italy

Ixodes ricinus from Italy were examined for the first time to detect whether rickettsiae were present. Using molecular methods, we detected three different spotted fever group rickettsiae, including Rickettsia helvetica. Our results raise the possibility that bacteria other than R. conorii are involved in rickettsial diseases in Italy.

Cockroaches that lack Blattabacterium endosymbionts: the phylogenetically divergent genus Nocticola

Phylogenetic relationships among termites, mantids and the five traditionally recognized cockroach families have been the subject of several studies during the last half-century. One cockroach lineage that has remained notably absent from such studies is the Nocticolidae. This group of small, elusive surface- and cave-dwelling species from the Old World Tropics has been proposed to represent an additional family. Using molecular sequences, we performed an initial phylogenetic examination of Nocticola spp. The hypothesis that they are phylogenetically divergent was confirmed from the analyses of three genes and a combined dataset. To supplement our phylogenetic analyses, we attempted to amplify 16S rRNA from the obligate mutualistic endosymbiont Blattabacterium cuenoti, present in all cockroaches studied to date. Unexpectedly, amplification was unsuccessful in all Nocticola spp. examined. This result was confirmed by microscopic examinations of fat body tissue. These Nocticola spp. are the first cockroaches found to be uninfected by B. cuenoti, which raise questions about when the bacterium first infected cockroaches.

Inhibition of the endosymbiont "Candidatus Midichloria mitochondrii" during 16S rRNA gene profiling reveals potential pathogens in Ixodes ticks from Australia.

BackgroundThe Australian paralysis tick (Ixodes holocyclus) is of significant medical and veterinary importance as a cause of dermatological and neurological disease, yet there is currently limited information about the bacterial communities harboured by these ticks and the risk of infectious disease transmission to humans and domestic animals. Ongoing controversy about the presence of Borrelia burgdorferi sensu lato (the aetiological agent of Lyme disease) in Australia increases the need to accurately identify and characterise bacteria harboured by I. holocyclus ticks.MethodsUniversal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16S rRNA genes present in DNA samples from I. holocyclus and I. ricinus ticks, collected in Australia and Germany respectively. The 16S amplicons were purified, sequenced on the Ion Torrent platform, and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomy. Initial analysis of I. holocyclus and I. ricinus identified that > 95 % of the 16S sequences recovered belonged to the tick intracellular endosymbiont “Candidatus Midichloria mitochondrii” (CMM). A CMM-specific blocking primer was designed that decreased CMM sequences by approximately 96 % in both tick species and significantly increased the total detectable bacterial diversity, allowing identification of medically important bacterial pathogens that were previously masked by CMM.ResultsBorrelia burgdorferi sensu lato was identified in German I. ricinus, but not in Australian I. holocyclus ticks. However, bacteria of medical significance were detected in I. holocyclus ticks, including a Borrelia relapsing fever group sp., Bartonella henselae, novel “Candidatus Neoehrlichia” spp., Clostridium histolyticum, Rickettsia spp., and Leptospira inadai.ConclusionsAbundant bacterial endosymbionts, such as CMM, limit the effectiveness of next-generation 16S bacterial community profiling in arthropods by masking less abundant bacteria, including pathogens. Specific blocking primers that inhibit endosymbiont 16S amplification during PCR are an effective way of reducing this limitation. Here, this strategy provided the first evidence of a relapsing fever Borrelia sp. and of novel “Candidatus Neoehrlichia” spp. in Australia. Our results raise new questions about tick-borne pathogens in I. holocyclus ticks.

Anaplasmataceae in wild rodents and roe deer from Trento Province (northern Italy)

In recent decades, a number of intracellular bacterial strains within the family Anaplasmataceae have been identified around the globe. These bacteria include Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis and Anaplasma marginale, which causes disease in ruminants. Bacteria from this family often have a wide range of hosts, infecting both vertebrates and invertebrates. A. phagocytophilum is an obligate intracellular pathogen that parasitises the granulocytes of humans and animals, such as domesticated dogs, sheep, cows and horses, as well as wildlife species, such as deer and rodents [1]. Various strains of A. phagocytophilum have been identified, but only some are considered human pathogens [2]. Different studies have demonstrated the role of the tick Ixodes ricinus as a potential vector for the transmission of A. phagocytophilum [1, 3, 4]. Since transovarial transmission of Anaplasma species appears to be inefficient in ticks, mammalian hosts are presumed to play an important role in the maintenance and propagation of Anaplasma species in nature [5]. However, the reservoir species of the human pathogenic strains of A. phagocytophilum in Europe is not known [2]. In Italy, the presence of members of the Anaplasmataceae family is known from screening studies of ticks; however, there are few data on their presence in vertebrate hosts. Human granulocytic anaplasmosis was first described in the USA in 1994 and is emerging in Europe [5, 6]. Although only three possible human cases have been reported in Italy [7, 8], serological and molecular studies have respectively shown A. phagocytophilum infections in domesticated animals and in I. ricinus ticks (see for example [3, 9]). To check the potential role of wild animals as reservoirs for these bacteria, we performed a PCR screening of either spleen or blood of roe deer (Capreolus capreolus) and bank voles (Clethrionomys glareolus) collected in Trento province, northern Italy. A total of 96 spleen samples collected in 2001 from roe deer that had either been hunted or found dead in Trento province were examined. Following DNA extraction using a DNeasy Tissue Kit (Qiagen, Hilden, Germany), PCR analysis with the primer pair PER1/PER2, which amplifies a 452-bp portion of the 16S rRNA of the Ehrlichia-Anaplasma group, was performed as reported previously [10]. PCR bands from the positive products were then sequenced directly. A total of 19 (19.8%) positive samples were found and then sequenced; the 452-bp sequences obtained from all samples were identical, revealing 100% identity with the 16S rRNA sequence of A. phagocytophilum (AF481853) previously reported in red deer, ticks and sheep. Following an identical procedure, 34 blood samples collected in 2002 from the bank vole C. glareolus were examined by PCR using the PER1/PER2 primer set. One positive result was found, and the sequence obtained revealed 100% identity with the 16S rRNA sequence of Candidatus Neoehrlichia mikurensis (AB213021) [11]. Eur J Clin Microbiol Infect Dis (2006) 25:677–678 DOI 10.1007/s10096-006-0196-x

Rickettsiae in Ixodid Ticks, Sicily

To the Editor: Members of the spotted fever group rickettsiae are intracellular bacteria usually associated with ixodid ticks, which are transferred to vertebrates by salivary secretions and within ticks transtadially and transovarially. Several tickborne rickettsiae cause human or animal diseases and, in the last 10 years, the increased use of molecular-based identification methods has resulted in new spotted fever group rickettsiae being characterized in ixodid ticks throughout Europe (1). Until recently, no rickettsiae, other than Rickettsia conorii, were reported in Italy. Since 2002, R. helvetica and Israeli spotted fever Rickettsia (R. conorii complex) have been detected in Ixodes ricinus and Rhipicephalus sanguineus, respectively (2–4). In Italy, Mediterranean spotted fever is endemic. This disease appears to occur more commonly in some central and southern regions (5); in 2002, more than half (498 of 890) of the cases of Mediterranean spotted fever identified in Italy and reported to the Ministry of Health came from Sicily.

Absence of the symbiont Candidatus Midichloria mitochondrii in the mitochondria of the tick Ixodes holocyclus

Candidatus Midichloria mitochondrii (M. mitochondrii) belongs to a novel clade of bacteria within the order Rickettsiales. Recent PCR-based screening studies indicate that it is present in a number of blood-sucking arthropods, as well as the blood of some vertebrates. Its medical and veterinary significance remains to be determined. Electron microscopic examinations of M. mitochondrii have thus far been conducted on two infected tick species. Remarkably, the bacterium was found in abundance within the mitochondria of the ovarian cells of each tick species. This makes it the only characterized bacterium able to invade the mitochondria of any multicellular organism. To examine whether mitochondrial invasion is a consistent characteristic of M. mitochondrii, we examined two tick species found in Eastern Australia. One of these species, Ixodes holocyclus, was infected with two M. mitochondrii strains; however, no bacteria were seen in the mitochondria. Comparative studies involving these strains may shed light on the unique phenomenon of mitochondrial invasion.

Molecular phylogeny of Cryptocercus wood-roaches based on mitochondrial COII and 16S sequences, and chromosome numbers in Palearctic representatives

Abstract Woodroaches of the genus Cryptocercus are subsocial and xylophagous cockroaches, distributed in North America and Asia. Studies on male chromosome number in Nearctic species have shown that diploid numbers vary from 2n=37 to 2n=47; numbers from Palearctic species were heretofore unknown. Two hypotheses have been proposed to explain the varying number of chromosomes among Nearctic species: the serial reduction hypothesis, and the parallel scenario. We performed phylogenetic analyses of the COII gene in these species and found evidence for the topology (47(45(43(39,37), which is congruent with the serial reduction hypothesis. We also determined chromosome numbers for the first time in Palearctic species, and found Cryptocercus primarius and Cryptocercus relictus to have relatively low chromosome numbers (2n=17–21) compared to their Nearctic relatives. Finally, our study determined the phylogenetic position of Cryptocercus primarius among other Asian taxa.

Cerebral Cryptococcomas in a Cow

Cerebral cryptococcomas are described in a 5-year-old mixed-breed cow without manifestations of systemic cryptococcosis. Two cryptococcomas were observed grossly. Microscopical examination revealed accumulations of yeast that were morphologically consistent with Cryptococcus neoformans. Immunohistochemistry characterized the organisms as C. neoformans var. grubii.