Candidatus Midichloria Research Articles

Molecular screening for Midichloria in hard and soft ticks reveals variable prevalence levels and bacterial loads in different tick species

Candidatus Midichloria mitochondrii, symbiont of the sheep tick Ixodes ricinus, was the first described member of the family Candidatus Midichloriaceae, order Rickettsiales. Recent reports are expanding our view of this family, now including numerous bacteria of great biological and medical interest, indicating a widespread distribution with an increasing range of hosts, with ticks being strongly represented.Here we present a molecular screening of 17 tick species, detecting and quantifying bacteria of the family Midichloriaceae in seven of them, including the first report of a representative of this family in a soft tick species (Argasidae), Ornithodoros maritimus. Based on sequence identity and phylogenetic analysis we propose that all these bacterial symbionts of ticks could be members of the genus Midichloria. The performed screening highlights different prevalence levels and variable bacterial loads in different tick species including one, Ixodes aulacodi, where the bacterium is present in all examined individuals, like in I. ricinus. This result prompts us to hypothesize different roles of Midichloria bacteria in different tick species.

Prevalence of pathogenic bacteria in Ixodes ricinus ticks in Central Bohemia.

Bacteria associated with the tick Ixodes ricinus were assessed in specimens unattached or attached to the skin of cats, dogs and humans, collected in the Czech Republic. The bacteria were detected by PCR in 97 of 142 pooled samples including 204 ticks, i.e. 1-7 ticks per sample, collected at the same time from one host. A fragment of the bacterial 16S rRNA gene was amplified, cloned and sequenced from 32 randomly selected samples. The most frequent sequences were those related to Candidatus Midichloria midichlori (71% of cloned sequences), followed by Diplorickettsia (13%), Spiroplasma (3%), Rickettsia (3%), Pasteurella (3%), Morganella (3%), Pseudomonas (2%), Bacillus (1%), Methylobacterium (1%) and Phyllobacterium (1%). The phylogenetic analysis of Spiroplasma 16S rRNA gene sequences showed two groups related to Spiroplasma eriocheiris and Spiroplasma melliferum, respectively. Using group-specific primers, the following potentially pathogenic bacteria were detected: Borellia (in 20% of the 142 samples), Rickettsia (12%), Spiroplasma (5%), Diplorickettsia (5%) and Anaplasma (2%). In total, 68% of I. ricinus samples (97/142) contained detectable bacteria and 13% contained two or more putative pathogenic groups. The prevalence of tick-borne bacteria was similar to the observations in other European countries.

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0958-3) contains supplementary material, which is available to authorized users.

Detection of tick-borne Anaplasma bovis, Anaplasma phagocytophilum and Anaplasma centrale in Spain.

The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes species of medical and veterinary importance. The presence of Anaplasma spp. in ticks from birds, as well as in Haemaphysalis punctata (Ixodida: Ixodidae) specimens collected from cattle and vegetation in northern Spain was investigated. A total of 336 ticks from birds [174 Ixodes frontalis (Ixodida: Ixodidae), 108 H. punctata, 34 Hyalomma marginatum (Ixodida: Ixodidae), 17 Ixodes ricinus (Ixodida: Ixodidae) and three Ixodes spp.], and 181 H. punctata specimens collected from cattle (n = 71) and vegetation (n = 110) were analysed. Anaplasma bovis was detected in five H. punctata, including two from birds (1.9%) and three from vegetation (2.7%). Four I. frontalis (2.3%) (one co-infected with 'Candidatus Midichloria mitochondrii') and one I. ricinus (5.9%) removed from birds, as well as four H. punctata (5.6%) collected from cattle showed Anaplasma phagocytophilum infection. In addition, Anaplasma centrale was found in two H. punctata, one from a cow (1.4%) and the other from vegetation (0.9%). This study represents the first evidence of the presence of A. bovis in European ticks, and reports the first detection of A. bovis and A. centrale in H. punctata, and the first finding of A. phagocytophilum and 'Ca. Midichloria mitochondrii' in I. frontalis.

Hard ticks and their bacterial endosymbionts (or would be pathogens)

The symbiotic microorganisms of arthropod vectors are highly significant from several points of view, partly due to their possible roles in the transmission of pathogenic causative agents by blood-sucking vectors. Although ticks are well studied because of their significance to human health, novel microbial associations remain to be described. This review summarises several endosymbiotic bacterial species in hard ticks from various parts of the world, including Coxiella-, Francisella-, Rickettsia- and Arsenophonus-like symbionts as well as Candidatus Midichloria mitochondrii and Wolbachia. New methodologies for the isolation and characterization of tick-associated bacteria will, in turn, encourage new strategies of tick control by studying their endosymbionts.

“Candidatus Defluviella procrastinata” and “Candidatus Cyrtobacter zanobii”, Two Novel Ciliate Endosymbionts Belonging to the “Midichloria Clade”

The "Midichloria clade" is a recently discovered but well-established evolutionary lineage clustering inside the order Rickettsiales (Alphaproteobacteria). Not much is known about the biology of these organisms. The best characterized ones are endocellular symbionts of very different eukaryotic hosts, ranging from arthropods to protists. "Candidatus Midichloria mitochondrii", the most studied organism of the group, is an interesting object of study because of its unique capability to infect metazoans' mitochondria and the presence of flagellar genes in its genome. With this work, we aim at increasing the knowledge on the biodiversity and phylogeny of the "Midichloria group". We characterized according to the "full cycle rRNA approach" two novel endosymbionts of ciliated protozoa, i.e. Paramecium nephridiatum and Euplotes aediculatus. According to the nomenclatural rules for uncultivated prokaryotes, we established the novel taxa "Candidatus Defluviella procrastinata" and "Candidatus Cyrtobacter zanobii" for the two bacterial symbionts. Our phylogenetic analysis based on 16S rRNA gene sequences confirms that the evolutionary histories of "Midichloria clade" representatives and of their hosts are very different. This suggests that the symbiotic processes arose many times independently, perhaps through ways of transmission still not described in Rickettsiales.

Screening for bacterial DNA in the hard tick Hyalomma marginatum (Ixodidae) from Socotra Island (Yemen): detection of Francisella-like endosymbiont

Thirty-four adult ticks collected from livestock on Socotra Island (Yemen) were identified as <em>Hyalomma marginatum</em> using traditional morphological characteristics. Morphological identification was confirmed for all the collected specimens using a molecular approach targeting a fragment of the mitochondrial gene 12S rRNA. All the specimens were examined for the presence of tick-borne pathogens and the tick endosymbiont <em>Candidatus </em>Midichloria mitochondrii using polymerase chain reaction. Three specimens out of the 34 analyzed tested positive to the presence of <em>Francisella </em>spp. leading to the first detection of these bacteria in <em>H. marginatum</em> on Socotra Island. The phylogenetic analyses conducted on a 660 bp fragment of the ribosomal gene 16S rRNA of <em>Francisella </em>spp. (including<em> F. philomiragia</em> as outgroup, the four subspecies of <em>F. tularensis</em> and the <em>Francisella</em>-like endosymbiont of ticks) confirm that the newly detected <em>Francisella </em>strains cluster into the <em>Francisella</em>-like endosymbionts of ticks. Interestingly, the detected <em>Francisella</em>-like endosymbiont, shows a different genotype to that previously isolated from <em>H. marginatum</em> collected in Bulgaria. No specimen was positive for the presence of <em>Rickettsia </em>spp., <em>Coxiella burnetii</em>, <em>Borrelia burgdorferi </em>or <em>M</em>. <em>mitochondrii</em>.

Localization of the bacterial symbiont Candidatus Midichloria mitochondrii within the hard tick Ixodes ricinus by whole-mount FISH staining

Here, we present an investigation on the spatial distribution of the bacterial symbiont Candidatus Midichloria mitochondrii within Ixodes ricinus, by whole mount fluorescence in situ hybridization (FISH). M. mitochondrii is a peculiar, recently discovered bacterium that resides in the mitochondria of female ticks. We applied a rapid and specific FISH protocol with oligonucleotide probes targeted on the 16S rRNA of M. mitochondrii, 12S rRNA of tick mitochondria, and a probe revealing active mitochondria. In this report that represents the first application of whole mount FISH on ticks, we observed strong, specific fluorescence signals in all the examined life stages, as the optimized protocol allowed us to overcome the autofluorescence interference of the cuticle. Cellular localization and quantification of the symbionts were also assessed with electron microscopy and specific real-time PCR, respectively.

Zoonotic Transmission of Pathogens byIxodes ricinusTicks, Romania

To the Editor: The Ixodes ricinus tick is a predominant vector of a large variety of pathogens of veterinary and medical consequence in Europe (1). The most prevalent I. ricinus–borne infection of persons in Europe is Lyme borreliosis, a multisystemic disorder caused by spirochetes of the Borrelia burgdorferi sensu lato complex (2). Persons bitten by ticks can also become infected with many other pathogens, such as bacteria (Anaplasma spp., Francisella spp., Coxiella burnetii, Bartonella spp., Rickettsiae spp., and Neoerhlichia mikurensis); parasites (Babesia spp., Theileria spp.); and arboviruses (tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, and Eyach virus) (1). Symptoms induced by such pathogens are often diverse and nonspecific, complicating accurate diagnosis of the disease. In Romania, cases of Lyme disease and tick-borne encephalitis (caused by tick-borne encephalitis virus) have been identified (3). However, little is known about the public health impact of these diseases, and none of the other tick-borne pathogens present in Europe have been reported as causes of infection in Romania. Although I. ricinus is the most abundant and widespread tick in Romania (3), the public health impact of I. ricinus–borne disease is likely to be underestimated. Therefore, the first step in evaluating the distribution of these potential pathogens is to establish their presence in ticks from previously unexplored areas. We conducted a study to identify the main tick-borne bacterial and parasitic human pathogens known to be present in Europe but not previously detected in Romania. We tested for the presence of DNA from spotted fever group Rickettsia spp., Anaplasma spp., Francisella tularensis, and Babesia spp. in 147 I. ricinus ticks collected from roe deer and goats at 2 sites in eastern Romania: Bacau (46°35′0″N, 26°55′0″E) and Galati (45°26′22″N, 28°2′4″E). Specimens were tested by PCR, using specific primers for each pathogen or group of pathogens, as described (4). Sequences obtained from Eurofins MWG Operon (Ebersberg, Germany) were identified by using BLAST (www.ncbi.nlm.nih.gov/BLAST) and compared with sequences available in GenBank. DNA from Rickettsia spp. was detected in 20 (13.6%) ticks. Sequence analyses revealed that 9 (6.1%) sequences were related to the R. monacensis strain IRd/Serbia glta gene (99%–100% nt similarity) (GenBank accession no. {type:entrez-nucleotide,attrs:{text:GQ925820,term_id:262181579,term_text:GQ925820}}GQ925820) and 11 (7.48%) were related to R. helvetica gltA gene (99%–100% nt similarity) (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AM418450,term_id:118918333,term_text:AM418450}}AM418450). DNA from Anaplasma spp. was identified in 33 (22.4%) ticks. Analysis revealed that 30 of the 33 amplified fragments showed 100% identity to the 16S rDNA gene of a symbiont in the family Anaplasmataceae, Candidatus Midichloria mitochondrii (GenBank accession no. {type:entrez-nucleotide,attrs:{text:EU780455,term_id:190607147,term_text:EU780455}}EU780455), and the remaining 3 were related to known pathogenic species identified in Romania: 2 (1.4%) exhibited 100% identity to Anaplasma phagocytophilum (GenBank accession no. {type:entrez-nucleotide,attrs:{text:EU982548,term_id:197360744,term_text:EU982548}}EU982548), and 1 (0.7%) showed 99% similarity to Ehrlichia muris (GenBank accession no. {type:entrez-nucleotide,attrs:{text:GU358691,term_id:284975291,term_text:GU358691}}GU358691). Francisella tularensis–specific DNA was amplified from 4 DNA extracts (2.7%). All 4 sequences were identical and shared 99% similarity with the F. tularensis peptidyl-propyl cis-trans isomerase gene (GenBank accession no. {type:entrez-nucleotide,attrs:{text:CP003048,term_id:377828067,term_text:CP003048}}CP003048). Babesia spp.–specific DNA was amplified in 1 DNA extract (0.7%), and it shared 99% sequence identity with the Babesia sp. EU1 18S rDNA gene (GenBank accession no. {type:entrez-nucleotide,attrs:{text:HQ830266,term_id:317383279,term_text:HQ830266}}HQ830266). This is a report on the identification of the human pathogens R. monacensis, R. helvetica, A. phagocytophilum, E. muris, and Babesia EU1 in Romania. R. monacensis (also known as the Cadiz agent or Rickettsia IRS3 and IRS4) was first identified in I. ricinus tick samples from Germany (5) and was recently recognized as the cause of Mediterranean spotted fever-like rickettsiosis in Spain (6). R. helvetica was isolated from I. ricinus ticks in Switzerland in 1979, and, since then, it has been isolated in many other European countries (7). R. helvetica was associated with human infections in the late 1990s (8). The role of animals as reservoirs for these pathogens is unknown. A. phagocytophilum is the causative agent of granulocytic anaplasmosis in humans, cattle, horses, and dogs, and is widespread throughout Europe (8). E. muris has been detected in ticks from Finland and European Russia and from ticks and rodents from Slovakia. In 2009, E. muris was also detected in patients with febrile illness in the United States (9). Babesia sp. EU1 has been detected in roe deer and ticks in several countries, and in 2003 was associated with human disease in Italy and Austria (4,10). Tularemia is known to be present in Romania and is thought to be exclusively and directly transmitted by hares. However, detection of F. tularensis DNA in I. ricinus ticks suggests that this bacterium might also be tick-borne. In conclusion, the detection of DNA of various human pathogens in ticks in Romania strongly suggests that these microorganisms circulate in the country. Because all of these pathogens affect humans, our study highlights the urgent requirement for further research to assess their impact on public health in Romania.

Multiple tick-associated bacteria in Ixodes ricinus from Slovakia

Eighty Ixodes ricinus ticks collected in the Rovinka forest in Slovakia were tested by real-time and routine single-step PCR for the presence of different pathogenic and endosymbiotic bacteria. No evidence of Coxiella burnetii, Diplorickettsia massiliensis, or Bartonella spp. was found. However, we identified, as the pathogenic bacteria, Anaplasma phagocytophilum in 8%, Rickettsia helvetica in 11%, Borrelia afzelii in 9%, Bo. garinii in 8%, Bo. burgdorferi sensu stricto in 5%, Bo. valaisiana in 3%, and Bo. miyamotoi in 1% of ticks. Other bacteria with unknown or suspected pathogenicity for humans were also identified: Arsenophonus nasoniae in 25%, Spiroplasma ixodetis in 3%, Candidatus Midichloria mitochondrii in 4%, Wolbachia aff. pipientis in 14%, and Ehrlichia muris in 3% of ticks. A. nasoniae was found almost exclusively in nymphs, and E. muris was only found in adult ticks. In total, 65% of ticks contained at least one species of bacteria, and 16% contained 2 or more species.

Presence, genetic variability, and potential significance of "Candidatus Midichloria mitochondrii" in the lone star tick Amblyomma americanum.

We used next generation sequencing to detect the bacterium "Candidatus Midichloria mitochondrii" for the first time in lone star ticks (Amblyomma americanum) from the eastern United States. 177 individuals and 11 tick pools from seven sites in four states were tested by pyrosequencing with barcoded 16S rRNA gene eubacterial primers targeting variable regions 5-3. Average infection prevalence was 0.15 across all surveyed populations (range 0-0.29) and only the site with the smallest sample size (n=5) was negative. Three genotypes differing by 2.6-4.1% in a 271bp region of 16S rRNA gene were identified. Two variants co-occurred in sites in North Carolina and New York, but were not observed in the same tick at those sites. The third genotype was found only in Georgia. Phylogenetic analysis of this fragment indicated that the three variants are more closely related to "Candidatus Midichloria mitochondrii" genotypes from other tick species than to each other. This variation suggests that multiple independent introductions occurred in A. americanum which may provide insight into bacterial spread within its ecosystem and parasitism on this tick. Whether the presence of this bacterium affects acquisition or maintenance of other pathogens and symbionts in A. americanum or the survival, biology and evolution of the tick itself is unknown.

A study on the presence of flagella in the order Rickettsiales: the case of ‘Candidatus Midichloria mitochondrii’

According to Bergey's Manual of Systematic Bacteriology, the Rickettsiales are '…bacteria with typical gram-negative cell walls and no flagella'. The recently sequenced genome of 'Candidatus Midichloria mitochondrii', a divergent lineage within the order Rickettsiales capable of invading mitochondria in ixodid ticks, revealed the presence of 26 putative flagellar genes. Open questions in relation to this observation are whether these genes are expressed and whether they possess the domains expected for the flagellar function. Here we show that: (a) the putative flagellar proteins of 'Ca. M. mitochondrii' actually possess the conserved domains and structural features required for their function in a model bacterium; (b) the seven flagellar genes of 'Ca. M. mitochondrii' that have been tested are expressed at the RNA level; and (c) the putative flagellar cap gene of this bacterium (FliD) is expressed at the protein level, and can be stained within the bacterium and at its surface. Beside the specific questions that we have addressed that relate to the first evidence, to our knowledge, for a flagellar apparatus in a member of the order Rickettsiales, we present here novel tools (recombinant protein and antibodies) that will facilitate the study of 'Ca. M. mitochondrii'.

Detection of bacteria related to Candidatus Midichloria mitochondrii in tick cell lines

Many ticks have been shown to be infected with intracellular bacteria. One of these bacteria is Candidatus Midichloria mitochondrii which is the only characterized bacterium that has the ability to invade the mitochondria within ovarian cells and consume them without any effect on the female tick's reproduction. In the present study, eight cell lines derived from the ticks Ixodes ricinus, Ixodes scapularis, Rhipicephalus (Boophilus) microplus, and Rhipicephalus (Boophilus) decoloratus were examined for the presence of the bacterium Ca. Midichloria mitochondrii. PCR assays for this bacterium were carried out using two sets of primers targeting the eubacterial 16SrRNA gene and a set of primers specific for the gyrB gene of Ca. Midichloria mitochondrii. With the 16S rRNA primers, DNA was amplified from two cell lines (R. (B.) decoloratus line BDE/CTVM14 and I. ricinus line IRE/CTVM19) on one out of three occasions each. Sequencing of the PCR products showed that the two cell lines gave sequences with 100% similarity to Ca. Midichloria mitochondrii. However, all cell lines, including the two positive cell lines, were negative with the specific primers. Phylogenetic analysis shows that our sequences belong to the subclass α-proteobacteria. They were identical to the sequences amplified from the tick I. ricinus. The results suggest that two cell lines, IRE/CTVM19 and BDE/CTVM14, may contain bacteria closely related to Ca. Midichloria mitochondrii and identical with it in a 350-bp part of the 16S rRNA gene sequence. To our knowledge, this constitutes the first report of the presence of DNA similar to the DNA of Ca. Midichloria mitochondrii in tick cell lines.

Phylogenomic Evidence for the Presence of a Flagellum and cbb3 Oxidase in the Free-Living Mitochondrial Ancestor

The initiation of the intracellular symbiosis that would give rise to mitochondria and eukaryotes was a major event in the history of life on earth. Hypotheses to explain eukaryogenesis fall into two broad and competing categories: those proposing that the host was a phagocytotic proto-eukaryote that preyed upon the free-living mitochondrial ancestor (hereafter FMA), and those proposing that the host was an archaebacterium that engaged in syntrophy with the FMA. Of key importance to these hypotheses are whether the FMA was motile or nonmotile, and the atmospheric conditions under which the FMA thrived. Reconstructions of the FMA based on genome content of Rickettsiales representatives-generally considered to be the closest living relatives of mitochondria-indicate that it was nonmotile and aerobic. We have sequenced the genome of Candidatus Midichloria mitochondrii, a novel and phylogenetically divergent member of the Rickettsiales. We found that it possesses unique gene sets found in no other Rickettsiales, including 26 genes associated with flagellar assembly, and a cbb(3)-type cytochrome oxidase. Phylogenomic analyses show that these genes were inherited in a vertical fashion from an ancestral α-proteobacterium, and indicate that the FMA possessed a flagellum, and could undergo oxidative phosphorylation under both aerobic and microoxic conditions. These results indicate that the FMA played a more active and potentially parasitic role in eukaryogenesis than currently appreciated and provide an explanation for how the symbiosis could have evolved under low levels of oxygen.

Absence of zoonotic Bartonella species in questing ticks: First detection of Bartonella clarridgeiae and Rickettsia felis in cat fleas in the Netherlands

BackgroundAwareness for flea- and tick-borne infections has grown in recent years and the range of microorganisms associated with these ectoparasites is rising. Bartonella henselae, the causative agent of Cat Scratch Disease, and other Bartonella species have been reported in fleas and ticks. The role of Ixodes ricinus ticks in the natural cycle of Bartonella spp. and the transmission of these bacteria to humans is unclear. Rickettsia spp. have also been reported from as well ticks as also from fleas. However, to date no flea-borne Rickettsia spp. were reported from the Netherlands. Here, the presence of Bartonellaceae and Rickettsiae in ectoparasites was investigated using molecular detection and identification on part of the gltA- and 16S rRNA-genes.ResultsThe zoonotic Bartonella clarridgeiae and Rickettsia felis were detected for the first time in Dutch cat fleas. B. henselae was found in cat fleas and B. schoenbuchensis in ticks and keds feeding on deer. Two Bartonella species, previously identified in rodents, were found in wild mice and their fleas. However, none of these microorganisms were found in 1719 questing Ixodes ricinus ticks. Notably, the gltA gene amplified from DNA lysates of approximately 10% of the questing nymph and adult ticks was similar to that of an uncultured Bartonella-related species found in other hard tick species. The gltA gene of this Bartonella-related species was also detected in questing larvae for which a 16S rRNA gene PCR also tested positive for "Candidatus Midichloria mitochondrii". The gltA-gene of the Bartonella-related species found in I. ricinus may therefore be from this endosymbiont.ConclusionsWe conclude that the risk of acquiring Cat Scratch Disease or a related bartonellosis from questing ticks in the Netherlands is negligible. On the other hand fleas and deer keds are probable vectors for associated Bartonella species between animals and might also transmit Bartonella spp. to humans.

Molecular detection of Ehrlichia canis, Anaplasma bovis, Anaplasma platys, Candidatus Midichloria mitochondrii and Babesia canis vogeli in ticks from Israel

Ticks are vectors of important pathogens of human and animals. Therefore, their microbial carriage capacity is constantly being investigated. The aim of this study was to characterize the diversity of domestic animal pathogens in ticks collected from vegetation and the ground, from different parts of Israel. Non-engorged questing adult ticks were collected from 13 localities. A total of 1196 ticks in 131 pools—83 pools of Rhipicephalus turanicus and 48 of Rhipicephalus sanguineus (with two to ten ticks per pool)—were included in this study. In addition, 13 single free-roaming Hyalomma spp. ticks were collected. Screening by molecular techniques revealed the presence of Ehrlichia canis, Anaplasma platys, Anaplasma bovis and Babesia canis vogeli DNA in R. turanicus ticks. E. canis, A. bovis, B. canis vogeli and Candidatus Midichloria mitochondrii DNA sequences were detected in R. sanguineus ticks. Candidatus Midichloria mitochondrii DNA was also detected in Hyalomma spp. ticks. Neither Hepatozoon spp. nor Bartonella spp. DNA was detected in any of the ticks examined. This study describes the first detection of E. canis in the tick R. turanicus, which may serve as a vector of this canine pathogen; E. canis was the most common pathogen detected in the collected questing ticks. It also describes the first detection of A. bovis and Candidatus Midichloria mitochondrii in Israel. To the best of the author's knowledge, this is the first report describing the detection of DNA of the latter two pathogens in R. sanguineus, and of A. bovis in R. turanicus.

Novel genotypes of Anaplasma bovis, “ Candidatus Midichloria” sp. and Ignatzschineria sp. in the Rocky Mountain wood tick, Dermacentor andersoni

Bovine anaplasmosis, caused by Anaplasma marginale, is a vector-borne disease that is enzootic in many parts of the USA. Although Dermacentor andersoni, a major vector of A. marginale, occurs in Canada, the Canadian cattle herds are currently considered free of bovine anaplasmosis. There have been two outbreaks of the disease in the province of Saskatchewan, but these have been linked to the importation of infected animals. However, the distribution of bovine anaplasmosis may alter with range expansion of the vectors. The aim of the present study was to use molecular techniques to determine if Anaplasma were present in D. andersoni at a locality near its northeastern distributional limit in Saskatchewan. Nested PCR analyses of the bacterial 16S rRNA gene were conducted on the total genomic DNA of 105 individual ticks. Single strand conformation polymorphism analysis and DNA sequencing of the 11 PCR-positive amplicons revealed the presence of three species of bacteria, none of which have been previously reported in D. andersoni. Although no ticks were infected with A. marginale, a novel genotype of A. bovis was detected in eight individuals. This discovery represents the first report of A. bovis in Canada. The potential implications of this finding with respect to animal health and anaplasmosis surveillance in Canada are discussed. The other two bacterial species detected were genetically similar to “ Candidatus Midichloria mitochondrii” and Ignatzschineria larvae, the latter of which has been associated with human disease in Europe. Further investigations are needed to determine the prevalence, reservoir hosts, and pathogenicity of the Canadian genotype of A. bovis.

“CandidatusAnadelfobacter veles” and “CandidatusCyrtobacter comes,” Two NewRickettsialesSpecies Hosted by the Protist CiliateEuplotes harpa(Ciliophora, Spirotrichea)

The order Rickettsiales (Alphaproteobacteria) is a well-known group containing obligate endocellular prokaryotes. The order encompasses three families (Rickettsiaceae, Anaplasmataceae, and Holosporaceae) and a fourth, family-level cluster, which includes only one candidate species, "Candidatus Midichloria mitochondrii," as well as several unnamed bacterial symbionts. The broad host range exhibited by the members of the "Candidatus Midichloria" clade suggests their eventual relevance for a better understanding of the evolution of symbiosis and host specificity of Rickettsiales. In this paper, two new bacteria belonging to the "Candidatus Midichloria" clade, hosted by two different strains of the ciliate protist Euplotes harpa, are described on the basis of ultrastructural observations, comparative 16S rRNA gene sequence analysis, and an estimation of the percentage of infection. Ultrastructure of these bacteria shows some unusual features: one has an electron-dense cytoplasm, and the other one lacks a symbiosomal membrane. The latter was up to now considered an exclusive feature of bacteria belonging to the family Rickettsiaceae. 16S rRNA gene phylogenetic analysis unambiguously places the new bacteria in the "Candidatus Midichloria" clade, although their phylogenetic relationships with other members of the clade are not clearly resolved. This is the first report of a ciliate-borne bacterium belonging to the "Candidatus Midichloria" clade. On the basis of the data obtained, the two bacteria are proposed as two new candidate genera and species, "Candidatus Anadelfobacter veles" and "Candidatus Cyrtobacter comes."

Absence of the symbiontCandidatusMidichloria mitochondrii in the mitochondria of the tickIxodes 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.

Detection of Alpha and Gamma-Proteobacteria in Amblyomma triste (Acari: Ixodidae) from Uruguay

Amblyomma triste is the most prevalent tick species reported in human tick bites in Uruguay and has been found to be infected with Rickettsia parkeri, but no other microorganisms have been reported from this tick. A sample of 254 adults of A. triste was collected by flagging on vegetation in suburban areas in southern Uruguay. Pools of five ticks were assembled and a screening for the DNA from the resulting 51 pools was realized by PCR assays using primers for amplifying a fragment of 16S rRNA gene for members of Anaplasmataceae. Seventeen pools were positive (33%) and the sequenciation of the gene fragment amplified revealed the presence of a putative new Alpha-Proteobacterium (denominated Atri-uru). The phylogenetic analysis showed that this microorganism is closely related to the symbiont of I. ricinus denominated 'Candidatus Midichloria mitochondrii' and other associated organisms. This rickettsial symbiont of ticks is included in a recent new clade proposed for the Alpha subclass of the Proteobacteria. The discovery of this bacterium in A. triste is the first evidence of this group of Rickettsiales detected in the Genus Amblyomma, and the first record in South America. Also, in two of 17 positive samples a Gamma-Proteobacterium related to Francisella-like organisms was detected.