Looking in ticks for human bacterial pathogens.

Microbial Pathogenesis xxx (2014) 1e7

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Looking in ticks for human bacterial pathogens O. Mediannikov a, b, *, F. Fenollar a, b a b

URMITE, UMR CNRS 7278 e IRD 198 e INSERM, Aix Marseille Universit e, Marseille, France Campus Universitaire IRD de Hann, Dakar, Senegal

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 September 2014 Accepted 12 September 2014 Available online xxx

Ticks are considered to be second worldwide to mosquitoes as vectors of human diseases and the most important vectors of disease-causing pathogens in domestic and wild animals. A number of emerging tick-borne pathogens are already discovered; however, the proportion of undiagnosed infectious diseases, especially in tropical regions, may suggest that there are still more pathogens associated with ticks. Moreover, the identification of bacteria associated with ticks may provide new tool for the control of ticks and tick-borne diseases. Described here molecular methods of screening of ticks, extensive use of modern culturomics approach, newly developed artificial media and different cell line cultures may significantly improve our knowledge about the ticks as the agents of human and animal pathology. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Ticks Ixodidae Rickettsia Borrelia Anaplasma Ehrlichia

1. Introduction Ticks are small arthropods in the order Ixodida, subclass Acarina. All ticks are ectoparasites highly specialized on hematophagy. Mammals, birds, reptiles and amphibians' blood is the only source of nutritional support of tick's body for both sexes and all developmental stages. Order Ixodida comprise three families: Ixodidae (hard ticks) with more than 700 officially recognized species [1], Argasidae (soft ticks) comprising roughly 200 species and Nuttalliellidae with a single species [2]. They occur worldwide and are capable of transmitting a broad range of human and animal pathogens including viruses, bacteria, protozoa and helminths, of which most have a life cycle that requires passage through the vertebrate host. Some of these pathogens are of exceptional importance because of high morbidity and mortality (in both humans and animals), long-lasting or permanent neuropsychiatric sequelae and impact on animal production [3,4]. 2. Identification of ticks The identification of tick species is a primary task in epidemiological studies of tick-borne diseases (Fig. 1). Morphological

de * Corresponding author. URMITE CNRS-IRD-INSERM UMR 7278 e Faculte decine, 27, Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. Me E-mail address: [email protected] (O. Mediannikov).

identification using standard taxonomic keys for endemic species in specific geographic regions is the oldest and very reliable approach. Since the birth of acarology, taxonomic keys were developed for specific regions of the world [5], hosts [6] and taxonomic phyla [7]. However, damaged ticks and immature stages may be very difficult to identify: the typical example is the ticks of Rhipicephalus (Boophilus) genus whose identification is based on the counting of the number of columns of the hypostomal teeth. Once the attached tick is removed leaving its hypostome inside the animal's skin, the morphological identification may be very difficult. Molecular methods were also developed to identify arthropods, including ticks. Mitochondrial markers (mitochondrial 12S and 16S ribosomal DNAs, cytochrome oxidase subunit 1 [COX1], cytochrome b) are usually easy to amplify and sequence, but the degree of intraspecies difference is rarely enough to distinguish the phylogenetically close species [8,9]. Nuclear markers (18S, 28S, internal transcribed spacers 1 and 2) have also been used, but only entire mitochondrial genome seems to be enough for phylogeneitc studies [10,11]. In addition to the technical and logistical drawbacks of PCR assays, this approach is further limited by the availability of gene sequences in genetic databases. Protein profiling by matrix-assisted laser desorption ionizationetime of flight mass spectrometry (MALDI-TOF MS) is now increasingly common for the routine identification of microorganisms in clinical microbiology [12]. In entomology and acarology, the MALDI-TOF MS approach was first applied to arthropods for the differentiation of Drosophila species [13]. Nowadays, this method,

http://dx.doi.org/10.1016/j.micpath.2014.09.008 0882-4010/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Mediannikov O, Fenollar F, Looking in ticks for human bacterial pathogens, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.09.008

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O. Mediannikov, F. Fenollar / Microbial Pathogenesis xxx (2014) 1e7

Fig. 1. Schema of identification of the tick species.

still developing, is quite promising as a cheap, rapid and quite reliable alternative for the identification of ticks [14,15]. 3. Research of pathogenic bacteria in ticks Identification of pathogenic bacteria in ticks using the approaches of molecular biology is one of the most often used and important tools in researches in infectious diseases. Sometimes, the search of certain pathogens in a tick may be important in clinical diagnostics, for instance, when the tick is removed from the patient. Identification of pathogenic bacteria (for instance, Lyme disease causing borreliae) in the ticks removed from humans may be necessary for the prophylactic antibiotic treatment to prevent Lyme disease [16,17]. However, mostly the research of pathogenic bacteria in ticks is performed in order to discover or precise the epidemiology of tickborne diseases including the identification of vectors and risk areas for certain bacteria. The identification of tick vectors may play important role in preventive measures against tick-borne diseases. 4. Molecular studies: general approach The first step of the identification of bacterial pathogens in ticks is usually based on PCR screening (Fig. 2). The choice of the primers used in the PCR entirely depends on the goals. In rare cases when the bacterial pathogen is well known and phylogenetically isolated as Coxiella burnetii, the screening with specific primers may be of use [18]. In many cases a bacterial genus contains many pathogens and the researcher may not know which exactly is the species that he or she may find in ticks. In such cases the approach include the screening with conventional or real-time PCR (qPCR) using the primers with broad specificity capable to produce the positive results in case of presence of all known (and even potentially unknown) species of the certain genus. It is the case of, for example genera Rickettsia, Borrelia and Bartonella. Further identification of

the exact species relies on either following species-specific (q)PCR or the amplification of the relatively large portion of a gene, its sequencing and the search for its identity in one of the genetic databases.

5. Group-specific and degenerate primers Standard PCR coupled with amplicon sequencing provide an invaluable tool for detection and identification of bacterial pathogens in any kind of sample, including ticks. Specificity of the reaction depends on complementarities of primers used in the PCR to the actual gene sequence of the researched bacteria. The specificity of primer(s) is a variable value that may be changed according to the goal of the research. Depending on the goals, sets of primers for conventional or qPCR previously reported may be used or may be designed. The scheme (Fig. 3) shows the common stages for the development of the broad range set of primers for the PCR. Briefly, the target gene should be quite conservative and be present in all organism of researched group (usually, housekeeping genes are good candidates). No genes with documented lateral gene transfers are accepted in order to evade the false positive results. The gene should contain the highly conservative sites in order to give the possibility to design the primers capable to hybridize with all or majority of bacteria from the group. However, a certain degree of variability is necessary; otherwise the BLAST search of the obtained sequence may not successfully identify the species. Optionally, the existing of the large database is quite important; otherwise the researcher should, however, identify and depose the sequences of most important species in the group in the genetic database. Growing number of the bacterial genome sequences available significantly facilitate the identification of bacteria. Once the gene is selected, the available sequences (often extracted from the genomes) are to be aligned with appropriate



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Fig. 2. Schema of the research of pathogens in ticks.

software (for example, ClustalW) and conservative sites for forward and reverse primers should be chosen. The distance between forward and reverse primers is to be approximately 400e700 base pairs because of the limitations of the direct amplicon sequencing resolution. If the researcher is designing the primers and probe for qPCR, the general rules are to be applied [19]. Sometimes the degenerate primers may be designed, usually because of the extensive variability of the selected gene. Degenerate primers are the mixtures of similar, but not identical primers. Differences among sequences are accounted for by using IUPAC degeneracies for individual bases. PCR primers are then synthesized as a mixture of primers corresponding to all permutations. Use of degenerate primers can greatly reduce the specificity of the PCR amplification. The problem can be partly solved by using touchdown PCR. Another advantage of the broad-range and degenerative primers is the eventual possibility to amplify the genes of yet unknown bacterium that may be pathogenic. Once designed, the specificity and sensitivity of primers are to be verified in silico (BLAST search) and in vitro (by testing the known positive controls). The first stage of screening of the DNA extracted from the tick (either whole or a part) for the important groups of pathogenic bacteria by (q)PCR (Fig. 2). The primers and probe sequences for qPCR used in our laboratory are presented in the Table 1. Most of the primers presented here were already used many times and proved its effectiveness not only to detect many representative of research group, but also to detect new species that predictably were amplified by group-specific/degenerate primers. For instance, in Sine-Saloum region of Senegal hard ticks collected from domestic animals were found to harbor at least five species of pathogenic rickettsiae: Rickettsia conorii conorii, Rickettsia africae, Rickettsia massiliae, Rickettsia aeschlimannii and Rickettsia sibirica mongolitimonae [20]. The initial screening of all ticks was

performed by qPCR aimed to amplify gltA (citrate synthase coding gene) of all rickettsia of spotted fever group. Then, taking in consideration the knowledge of the distribution of spotted fever rickettsiae on African continent, the qPCR specific for R. africae [21] and R. aeschlimannii [20] were used to identify these rickettsiae among the positive samples when tested by the first groupspecific gltA-based qPCR. Those samples that were positive by “all rickettsia” qPCR but negative by R. africae- and R. aeschlimanniispecific qPCR were subjected to amplification followed by sequencing of the “reference” genes, for Rickettsia it is almost entire size gltA gene. Nucleotide BLAST [22] allowed to identify the rickettsia. 6. Validation of molecular studies The presence of appropriate positive and negative controls in each PCR assay is necessary to validate the assay. The use of positive controls (bacterial DNA) allows to validate the PCR itself. DNA from a microorganism which was never been reported from ticks or in specific region is preferred instead of a common causative pathogen. For example, Rickettsia montanensis may be used as a positive control when European or African ticks are studies. The use of negative controls, processed from DNA extraction to PCR in parallel to the tested samples, is imperative to detect contamination. Water, a mixture of all reagents used in the PCR assay and DNA extracted from human tissue without infection can be used as negative controls [23]. 7. Microbiome studies Tick microbiomes remain up to date largely unexplored. The first study by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing technique to characterize bacterial diversity of Rhipicephalus (Boophilus) microplus appeared in 2011 [24]. This study revealed the presence of bacteria of 121 genera,


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Fig. 3. Schema of the selection of candidate sites for the group-specific oligonucleotides.

including Borrelia, Coxiella, Wolbachia and Legionella. Among others, microbiomes of Ixodes ricinus [25], Amblyomma testudinarium, Amblyomma variegatum, Haemaphysalis formosensis, Haemaphysalis longicornis, Ixodes ovatus, Ixodes persulcatus [26], Amblyomma americanum [27], and Amblyomma maculatum [28] were analyzed. 454- and Illumina-based metagenomic approaches were used along with pyrosequencing of bacteriaenriched whole genome or prokaryotic 16S rRNA. Studies of tick microbiomes are the most extensive approach for the studies of bacteria associated with ticks and the most powerful tool for the identification of new bacteria in ticks. Unfortunately, the methods stay quite expensive and up to date few tick species were studied. In spite of the paucity of available data, the number of bacterial genera (more than 100 in almost all studies) identified in ticks is astonishing. However, most of the identified genera contain

environmental bacteria never reported to be associated with arthropods or pathology of the vertebrates. However, bacteria from the genera Borrelia, Rickettsia, Candidatus Neoehrlichia, Wolbachia, Anaplasma, Ehrlichia, Candidatus Midichloria mitochondrii, Francisella, Rickettsiella, Coxiella, Bartonella, and Chlamydia were repeatedly reported from ticks. Interestingly, some species attributed to these genera are evidently new species that were never identified in ticks before. 8. Research of pathogenic bacteria: biological studies Microbial culture is one of the primary diagnostic methods of microbiology and used as a tool to determine the cause of infectious disease and potential pathogenicity by letting the agent multiply in a predetermined medium. Microbial cultures are foundational and



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Table 1 List of primers. Specificity

Use

Gene target

Name of the oligonucleotidea

Oligonucleotide sequences

Reference

Rickettsia genusspecific

Screening for Rickettsia spp.

gltA

RKND03_F RKND03_R RKND03_P

[20]

Rickettsia genusspecific

Confirmation

Hypothetical protein

Rick_1029_F1 Rick_1029_R1 Rick_1029_P

Anaplasmataceae

Screening for Anaplasma and Ehrlichia spp.

23S

TtAna-f TtAna-r TtAna-s

Borrelia genus specific Screening for Borrelia spp.

16S

Bor_16S_3_F Bor_16S_3_R Bor_16S_3_P

Bartonella genus specific

Screening

16Se23S ITS

Barto_ITS3_F Barto_ITS3_R Barto_ITS3_P

Confirmation

16Se23S ITS

Barto_ITS2_F Barto_ITS2_R Barto_ITS2_P

Group of Coxiella-like bacteria

Screening

16S

Coxlike-Rh_F Coxlike-Rh_R Coxlike-Rh_L

Coxiella burnetii

Screening, specific search

IS1111A

IS1111_F IS1111_F IS1111_P

confirmation

Hypothetical protein

IS30A_F IS30A_F IS30A_F

Arsenophonus nasoniae Specific search

ftsY

Arsftsf ArsftsYr ArsftsYp

Spiroplasma aff. ixodetis

rpoB

rpoBf rpoBr rpoBp

50 -GTGAATGAAAGATTACACTATTTAT-30 50 -GTATCTTAGCAATCATTCTAATAGC-30 6FAM- CTATTATGCTTGCGGCTGTCGGTTCTAMRA 50 -GAMAAATGAATTATATACGCCGCAAA30 50 -ATTATTKCCAAATATTCGTCCTGTAC-30 6FAMCGGCAGGTAAGKATGCTACTCAAGATAATAMRA 50 -TGACAGCGTACCTTTTGCAT-30 50 -TGGAGGACCGAACCTGTTAC-30 6FAM-GGATTAGACCCGAAACCAAGTAMRA 50 -AGCCTTTAAAGCTTCGCTTGTAG-30 50 -GCCTCCCGTAGGAGTCTGG-30 6FAM- CCGGCCTGAGAGGGTGAACGGTAMRA 50 -GATGCCGGGGAAGGTTTTC-30 50 -GCCTGGGAGGACTTGAACCT-30 6FAM- GCGCGCGCTTGATAAGCGTGTAMRA 50 -GGGGCCGTAGCTCAGCTG-30 50 -TGAATATATCTTCTCTTCACAATTTC-30 6FAM- CGATCCCGTCCGGCTCCACCATAMRA 50 -ACCTACCCTTGACATCCTCGGAA-30 50 -GCAACTAAGGACGAGGGTTG-30 6FAM- CAGCTCGTGTCGTGAGATGTTAMRA 50 -CAAGAAACGTATCGCTGTGGC-30 50 -CACAGAGCCACCGTATGAATC-30 6FAM- CCGAGTTCGAAACAATGAGGGCTGTAMRA 50 -CGCTGACCTACAGAAATATGTCC-30 50 -GGGGTAAGTAAATAATACCTTCTGG-30 6FAMCATGAAGCGATTTATCAATACGTGTATGCTAMRA 50 -TGGGGTGGGTAAAACCACTA-30 50 -TTGTTACGCTCTCCCCAAAC-30 6-FAM-TTAGCCCGTCAATATCAGGCTAMRA 50 -TGTTGGACCAAACGAAGTTG-30 50 -CCAACAATTGGTGTTTGTGG-30 6-FAM-GCTAACCGTGCTTTAATGGGTAMRA

a

Specific search

[20]

Personal communications

[50]

[51]

Personal communications

[18]

[47]

[47]

F for forward, R for reverse, P for probe.

basic diagnostic methods used extensively as a research tool in molecular biology. It is essential for the official description of the new taxons. In case of tick-associated bacteria, all of pathogenic bacteria are fastidious and difficult to isolate in axenic culture. Many are obligate intracellular bacteria as Rickettsia, Anaplasma, Ehrlichia and Coxiella [29]. All Borrelia spp. require the complex artificial media, frequent subcultures and strict temperature regime [14]. Bartonella spp. are very slow-growing bacteria that require a rich specific medium supplied with erythrocytes [30]. Many bacterial species have not yet been ever isolated in pure culture and since were not yet officially described. Often these bacteria have the Candidatus status [31] as Candidatus Neoehrlichia mikuriensis or Candidatus Midichloria mitochondrii or just a surname as Coxiella-like bacteria or Francisella-like bacteria [32,33]. The identification of isolated bacteria is usually done by common methods: molecular identification (amplification and sequencing of 16S rRNA gene and other genes accepted for the characterization of specific groups of bacteria) is the gold standard

for the identification. Most of the new species discovered in ticks originate from occasional bacterial culture. 8.1. Microbial culture on artificial media The traditional approach in bacteriology is using artificial media jellified with agarose (agar). This approach is, however, not very interesting in studies of bacterial diversity of ticks, because almost all of bacterial colonies obtained like this are environmental bacteria most probably originating from the tick's surface [34]. However, in several cases the isolation of Bartonella species may be achieved [18,35]. 8.2. Animal models Laboratory animals may be also used for the isolation of bacteria in pure culture. In biology, axenic describes the state of a culture in which only a single species, variety, or strain of organism is present and entirely free of all other contaminating organisms. So neither


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the bacterial strain isolated with an animal model nor bacterial strain isolated in the cell culture (see below) cannot be called “axenic” sensu stricto. However, in both cases the animal and the cells are counted for the “medium” or “environment” for the bacteria and not for the separate organism, so the isolation of bacteria using animal models and cell lines is an accepted method in bacteriology [36]. Animals are traditionally used for the isolation of such tickassociated bacteria as Rickettsia spp [37]. and Borrelia spp [38]. White mice may also be used as a sentinel animals: leaving the cages with white mice in the areas endemic for borreliosis, mice may be naturally infected by the infected tick bites [39]. However, due to the development of a new artificial media for Borrelia [38] and cell culture for Rickettsia [40] and some ethical issues, using the laboratory animals for the isolation of pathogenic bacteria from tick is not widely practiced. 8.3. Cell culture Such bacterial genera associated with ticks as Rickettsia, Anaplasma, Ehrlichia, Coxiella, Wolbachia, Neorickettsia, Candidati Neoehrlichia and Midichloria, are obligate intracellular. Several successful attempts have been performed in order to develop the artificial medium for the culture of Rickettsia and Coxiella [41,42], however, the cell culture is still the method of choice for its isolation. Isolation of bacterial using the cell culture is a laboratory test in which samples are placed with a cell type that the bacteria being tested for is able to infect. The method is the same that is extensively used in virology. Two major differences are 1) using of antibiotic is restricted in order to not inhibit the growth of bacteria and 2) the bacterial colonization of cells does not necessarily produce the cytopathic effect. The identification of the successful isolation of intracellular bacteria lies on either specific coloration methods developed for intracellular bacteria [43,44] or on the methods of molecular identification. The search of the bacterial pathogens in ticks lies often on the blind inoculation of triturated tick in the one or several cell line cultures and the following screening of the cell cultures for the presence of bacteria. Quite often in spite of the precautions the culture is contaminated by environmental bacterial flora, so thorough washing and disinfection of ticks is an important issue for the successful culture. The screening is often done by the coloration of the cells by Gimenez [44] staining or other methods (DiffQuick, Gram). 8.4. Choice of the cell lines The sensitivity of the specific cell line for the specific bacterium is often the key issue for the successful isolation. Cell lines most often used for the isolation of intracellular bacteria are presented in the Table 2. Methodically, it may be quite important to use several cell lines for the inoculation of the same samples. 8.5. Combined methods For some bacteria the combined methods were used for the isolation. Some rickettsial species were isolated in pure culture by initial inoculation in animals (that in this case were used as the enrichment and purification medium) followed by the re-isolation in cell culture [37]. Arsenophonus nasoniae was firstly isolated in the cell culture and then transferred to agar plate cultures [45]. Spiroplasma ixodetis was initially isolated from Ixodes pacificus ticks using the artificial medium [46], the strain isolated from I. ricinus, however, was cultivated only in co-culture with XTC-2 cells [47].

Table 2 Several examples of the cell lines and their usage for the isolation of bacterial strains from ticks. Cell line

Origin

Bacteria isolated using this cell line References

XTC-2 Xenopus laevis

[47,52,53]

L929

[54]

Vero

C6/36 ISE6 BME/

Diplorickettsia massiliensis, Spiroplasma ixodetis, most of the rickettsiae including Rickettsia helvetica Mouse fibroblasts Most of the rickettsiae including Rickettsia aeschlimannii Kidney epithelial cells Most of the rickettsiae including from an African green Rickettsia africae, Coxiella burnetii monkey Aedes albopictus larvae Rickettsia hoogstraalii Wolbachia pipientis Ixodes scapularis eggs Anaplasma phagocytophilum CTVM2 Rhipicephalus (Boophilus) microplus eggs

[59] HL-60 Human promyelocytic Anaplasma phagocytophilum leukemia cells DH82 Canine monicyte Ehrlichia chaffeensis, Ehrlichia macrophages ruminantium

[55]

[56,57] [58] Rickettsia raoultii [60] [61,62]

9. Perspectives Ticks are considered to be second worldwide to mosquitoes as vectors of human diseases and the most important vectors of disease-causing pathogens in domestic and wild animals. Continuing discoveries of emerging tick-borne pathogens and the number of undiagnosed infectious diseases, especially in tropical regions, may suggest that there are still new pathogens associated with ticks to be discovered. Moreover, the identification of bacteria associated with ticks may provide new tool for the control of ticks and tick-borne diseases. Molecular methods of screening of ticks, extensive use of modern culturomics approach [48], newly developed artificial media [41,49] and different cell line cultures may significantly improve our knowledge about the ticks as the agents of human and animal pathology. References [1] Guglielmone AA, Robbins RG, Apanaskevich DA, Petney TN, Estrada-Pena A, Horak IG. In: Springer, editor. The Hard Ticks of the World (Acari: Ixodida: Ixodidae). New York e London: Springer; 2014. [2] Guglielmone AA, Robbins RG, Apanaskevich DA, Petney TN, Estrada-Pena A, Horak IG, et al. The Argasidae, Ixodidae and Nuttalliellidae (Acari: Ixodida) of the world: a list of valid species names. Zootaxa 2010;2528:1e28. [3] Brown CG. Dynamics and impact of tick-borne diseases of cattle. Trop. Anim. Health Prod. 1997 Nov;29(4 Suppl. l):1Se3S. [4] Baneth G. Tick-borne infections of animals and humans: a common ground. Int. J. Parasitol. 2014 Aug;44(9):591e6. [5] Hoogstraal H. In: African Ixodoidea I. Ticks of the Sudan (With Special Reference to Equatoria Province and with Preliminary Reviews of the Genera Boophilus, Margaropus, and Hyalomma. Washington, D.C: U. S. Navy, Washington, D. C.; 1956. [6] Walker AR, Bouattour A, Camicas JL, Estrada-Pena A, Horak IG, Latif AA, et al. Ticks of Domestic Animals in Africa. Edinburgh, UK: Bioscience Reports; 2003. [7] Walker JB, Keirans JE, Horak IG. The Genus Rhipicephalus (Acari, Ixodidae). A Guide to the Brown Ticks of the World. New York: Cambridge University Press; 2000. [8] Norris DE, Klompen JS, Keirans JE, Black WC. Population genetics of Ixodes scapularis (Acari: Ixodidae) based on mitochondrial 16S and 12S genes. J. Med. Entomol. 1996 Jan;33(1):78e89. [9] Song S, Shao R, Atwell R, Barker S, Vankan D. Phylogenetic and phylogeographic relationships in Ixodes holocyclus and Ixodes cornuatus (Acari: Ixodidae) inferred from COX1 and ITS2 sequences. Int. J. Parasitol. 2011 Jul;41(8): 871e80. [10] Burger TD, Shao R, Labruna MB, Barker SC. Molecular phylogeny of soft ticks (Ixodida: Argasidae) inferred from mitochondrial genome and nuclear rRNA sequences. Ticks Tick. Borne Dis. 2014 Mar;5(2):195e207. [11] Burger TD, Shao R, Beati L, Miller H, Barker SC. Phylogenetic analysis of ticks (Acari: Ixodida) using mitochondrial genomes and nuclear rRNA genes



[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19] [20]

[21] [22] [23] [24]

[25]

[26]

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[28]

[29] [30]

[31]

[32]

[33]

[34] [35]

[36]

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Pathogens of Acarina (ticks).

Looking in apes as a source of human pathogens.

Prevalence of vector-borne bacterial pathogens in riparian brush rabbits (Sylvilagus bachmani riparius) and their ticks.

Molecular detection of bacterial and parasitic pathogens in hard ticks from Portugal.

Plant innate immunity against human bacterial pathogens.

Microbial population analysis of the salivary glands of ticks; a possible strategy for the surveillance of bacterial pathogens.

Brandenburg, Germany.

Examination of faeces for bacterial pathogens.

Ticks and associated pathogens in dogs from Greece.

Examination of faeces for bacterial pathogens.

Heme uptake in bacterial pathogens.

Human mesenchymal stem cells: New sojourn of bacterial pathogens.

Molecular analysis for screening human bacterial pathogens in municipal wastewater treatment and reuse.

How old are bacterial pathogens?

Bacterial Profiling Reveals Novel "Ca. Neoehrlichia", Ehrlichia, and Anaplasma Species in Australian Human-Biting Ticks.

Bacterial pathogens of Culicidae (mosquitos).

Host specificity of bacterial pathogens.

Heme Synthesis and Acquisition in Bacterial Pathogens.

Chronic Bacterial Pathogens: Mechanisms of Persistence.

Cytoplasmic access by intracellular bacterial pathogens.

Looking back, looking forward: 55 years of human factors.

Bacterial pathogens associated with hidradenitis suppurativa, France.

The importance of the viable but non-culturable state in human bacterial pathogens.

Tick infestation in birds and prevalence of pathogens in ticks collected from different places in Germany.