Prevalence of Tick-Borne Pathogens from Ticks Collected from Cattle and Wild Animals in Tanzania in 2012.

This study was aimed to disclose the prevalence rate of tick-borne pathogens from ticks collected from cattle and wild animals in Tanzania in 2012. Ticks were collected from slaughtered cattle and dead wild animals from November 5 to December 23, 2012 and identified. PCR for detecting Anaplasmataceae, Piroplamidae, Rickettsiaceae, Borrelia spp., and Coxiella spp. were done. Among those tested, Rickettsiaceae, Piroplasmidae, and Anaplasmataceae, were detected in ticks from the 2 regions. Rickettsiaceae represented the major tick-borne pathogens of the 2 regions. Ticks from animals in Maswa were associated with a higher pathogen detection rate compared to that in ticks from Iringa. In addition, a higher pathogen detection rate was observed in ticks infesting cattle than in ticks infesting wild animals. All examined ticks of the genus Amblyomma were infected with diverse pathogens. Ticks of the genera Rhipicephalus and Hyalomma were infected with 1 or 2 pathogens. Collectively, this study provides important information regarding differences in pathogen status among various regions, hosts, and tick species in Tanzania. Results in this study will affect the programs to prevent tick-borne diseases (TBD) of humans and livestock in Tanzania.

Hijacking of Membrane Contact Sites by Intracellular Bacterial Pathogens.

Intracellular bacterial pathogens have evolved sophisticated mechanisms to hijack host cellular processes to promote their survival and replication inside host cells. Over the past two decades, much attention has been given to the strategies employed by these pathogens to manipulate various vesicular trafficking pathways. But in the past 5 years, studies have brought to light that intracellular bacteria also target non-vesicular trafficking pathways. Here we review how three vacuolar pathogens, namely, Legionella, Chlamydia, and Coxiella hijack components of cellular MCS with or without the formation of stable MCS. A common theme in the manipulation of MCS by intracellular bacteria is the dependence on the secretion of bacterial effector proteins. During the early stages of the Legionella life cycle, the bacteria connects otherwise unrelated cellular pathways (i.e., components of ER-PM MCS, PI4KIIIα, and Sac1 and the early secretory pathway) to remodel its nascent vacuole into an ER-like compartment. Chlamydia and Coxiella vacuoles establish direct MCS with the ER and target lipid transfer proteins that contain a FFAT motif, CERT, and ORP1L, respectively, suggesting a common mechanism of VAP-dependent lipid acquisition. Chlamydia also recruits STIM1, an ER calcium sensor involved in store-operated calcium entry (SOCE) at ER-PM MCS, and elucidating the role of STIM1 at ER-Chlamydia inclusion MCS may uncover additional role for these contacts. Altogether, the manipulation of MCS by intracellular bacterial pathogens has open a new and exciting area of research to investigate the molecular mechanisms supporting pathogenesis.

Diversity of Coxiella-like and Francisella-like endosymbionts, and Rickettsia spp., Coxiella burnetii as pathogens in the tick populations of Slovakia, Central Europe.

Ticks are important vectors of pathogens affecting humans and animals worldwide. They do not only carry pathogens but diverse commensal and symbiotic microorganisms are also present in ticks. A molecular screening for tick-borne pathogens and endosymbionts was carried out in Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis inermis questing ticks collected in Slovakia. The presence of Rickettsia spp., Coxiella burnetii, Coxiella-like and Francisella-like microorganisms was evaluated by PCR in 605 individuals and by randomly sequencing 66 samples. Four species of rickettsiae (R. raoultii, R. slovaca, R. helvetica and R. monacensis) were identified and reported with an overall prevalence range between 0.4 and 50.3% (±8.0) depending on tick species, sex and locality. Partial sequencing of the gltA gene of 5 chosen samples in H. inermis showed 99% identity with Candidatus Rickettsia hungarica. The total prevalence of C. burnetii in ticks was 2.2 ±â€¯1.7%; bacteria were confirmed in I. ricinus and D. reticulatus ticks. The sequences from 2 D. reticulatus males and 1 I. ricinus female ticks were compared to GenBank submissions and a 99.8% match was obtained with the pathogenic C. burnetii. Coxiella-like endosymbionts were registered in all three species of ticks from all studied sites with an average prevalence of 32.7 ±â€¯3.7%. A phylogenetic analysis of this Coxiella sp. showed that it does not group with the pathogenic C. burnetii. The prevalence of Francisella-like microorganisms in questing ticks was 47.9 ±â€¯3.9%, however H. inermis (n = 108) were not infested. Obtained sequences were 98% identical with previously identified Francisella-like endosymbionts in D. reticulatus and I. ricinus. Coxiella-like and Francisella-like microorganisms were identified for the first time in Slovakia, they might be considered as a non-pathogenic endosymbiont of I. ricinus, D. reticulatus and H. inermis, and future investigations could aim to assess their role in these ticks. However, this work provided further data and broadened our knowledge on bacterial pathogens and endosymbionts present in ticks in Slovakia to help understanding co-infestations, combined treatments and public health issues linked to tick bites.

Antimicrobial Resistance in Chlamydiales, Rickettsia, Coxiella, and Other Intracellular Pathogens.

This article will provide current insights into antimicrobial susceptibilities and resistance of an important group of bacterial pathogens that are not phylogenetically related but share lifestyle similarities in that they are generally considered to be obligate intracellular microbes. As such, there are shared challenges regarding methods for their detection and subsequent clinical management. Similarly, from the laboratory perspective, susceptibility testing is rarely undertaken, though molecular approaches might provide new insights. One should also bear in mind that the highly specialized microbial lifestyle restricts the opportunity for lateral gene transfer and, consequently, acquisition of resistance.

Manipulation of Host Cholesterol by Obligate Intracellular Bacteria.

Cholesterol is a multifunctional lipid that plays important metabolic and structural roles in the eukaryotic cell. Despite having diverse lifestyles, the obligate intracellular bacterial pathogens Chlamydia, Coxiella, Anaplasma, Ehrlichia, and Rickettsia all target cholesterol during host cell colonization as a potential source of membrane, as well as a means to manipulate host cell signaling and trafficking. To promote host cell entry, these pathogens utilize cholesterol-rich microdomains known as lipid rafts, which serve as organizational and functional platforms for host signaling pathways involved in phagocytosis. Once a pathogen gains entrance to the intracellular space, it can manipulate host cholesterol trafficking pathways to access nutrient-rich vesicles or acquire membrane components for the bacteria or bacteria-containing vacuole. To acquire cholesterol, these pathogens specifically target host cholesterol metabolism, uptake, efflux, and storage. In this review, we examine the strategies obligate intracellular bacterial pathogens employ to manipulate cholesterol during host cell colonization. Understanding how obligate intracellular pathogens target and use host cholesterol provides critical insight into the host-pathogen relationship.

The diverse habitats of obligate intracellular parasites.

Bacterial obligate intracellular parasites have evolved diverse mechanisms for evasion of host cellular defenses. These mechanisms involve adaptations for survival in distinct intracellular compartments. Intracellular niches inhabited by obligate intracellular parasites include the cytoplasm, arrested early endosomes, lysosomes, and vesicles that do not fuse with the endosomal compartment but intersect with an exocytic pathway.

Subversion of Retrograde Trafficking by Translocated Pathogen Effectors.

Intracellular bacterial pathogens subvert the endocytic bactericidal pathway to form specific replication-permissive compartments termed pathogen vacuoles or inclusions. To this end, the pathogens employ type III or type IV secretion systems, which translocate dozens, if not hundreds, of different effector proteins into their host cells, where they manipulate vesicle trafficking and signaling pathways in favor of the intruders. While the distinct cocktail of effectors defines the specific processes by which a pathogen vacuole is formed, the different pathogens commonly target certain vesicle trafficking routes, including the endocytic or secretory pathway. Recently, the retrograde transport pathway from endosomal compartments to the trans-Golgi network emerged as an important route affecting pathogen vacuole formation. Here, we review current insight into the host cell's retrograde trafficking pathway and how vacuolar pathogens of the genera Legionella, Coxiella, Salmonella, Chlamydia, and Simkania employ mechanistically distinct strategies to subvert this pathway, thus promoting intracellular survival and replication.

Analysis of the cbhE' plasmid gene from acute disease-causing isolates of Coxiella burnetii.

A gene termed cbhE' was cloned from the QpH1 plasmid of Coxiella burnetii. Expression of recombinants containing cbhE' in vitro and in Escherichia coli maxicells, produced an insert-encoded polypeptide of approx. 42 kDa. The CbhE protein was not cleaved when intact maxicells were treated with trypsin. Hybridizations of total DNA isolated from the six strains of C. burnetii indicate that this gene is unique to C. burnetii strains associated with acute disease, i.e., Hamilton[I], Vacca[II], and Rasche[III]. The cbhE' gene was not detected in strains associated with chronic disease (Biotzere[IV] and Corazon[V]) or the Dod[VI] strain. The cbhE' open reading frame (ORF) is 1022 bp in length and is preceded by a predicted promoter/Shine-Dalgarno (SD) region of TCAACT(-35)-N16-TAAAAT(-10)-N14-AGAAGGA (SD) located 10 nucleotides (nt) before the presumed AUG start codon. The ORF ends with a single UAA stop codon and has no apparent Rho-factor-independent terminator following it. The cbhE' gene codes for the CbhE protein of 341 amino acid (aa) residues with a deduced Mr of 39,442. CbhE is predominantly hydrophilic with a predicted pI of 4.43. The function of CbhE is unknown. No nt or aa sequences with homology to cbhE' or CbhE, respectively, were found in searches of a number of data bases.

Coinfection of fibroblasts with Coxiella burnetti and Toxoplasma gondii: to each their own.

Intracellular pathogens have evolved distinct strategies to subvert host cell defenses. At diametrically opposed ends of the spectrum with regard to the host endosomal/lysosomal defenses are the obligate intracellular protozoan Toxoplasma gondii and the bacterium Coxiella burnetti. While the intracellular replication of T. gondii requires complete avoidance of the host endocytic cascade, C. burnetti actively subverts it. This results in these organisms establishing and growing in very different vacuolar compartments. In this study we examined the potential interaction between these distinct compartments following coinfection of mammalian fibroblasts. When present within the same cell, these organisms exhibit minimal interaction with each other. Colocalization of T. gondii and C. burnetti within the same vacuole occurs at a low frequency in doubly infected cells. In such instances only one of the organisms appears to be replication competent, emphasizing the different requirements for survival and/or intracellular growth. The potential basis for both the lack of interaction between these distinct pathogen-containing compartments, and the mechanisms to address their low frequency of colocalization are discussed in the context of our understanding of the biology of the organisms and membrane traffic in eukaryotic cells.

The biology of Coxiella burnetti and the pathobiochemistry of Q fever and its endotoxicosis.

C. burnetii possesses a battery of host-independent enzymes which mediate endergonic and exergonic reactions. The biochemical and biophysical lesions responsible for the organism's obligate intracellular parasitic state have not been identified. Clues to this fundamental problem may lie in the agent's acidophilic metabolism and proliferation within the host cell's phagolysomal vacuole. What are the modifiers of transcription and translation? What constituents of the lysosomal vacuole contribute to the parasite's metabolism? Does the parasite have intrinsic cell-wall synthesizing capability? The isolation of plasmids from C. burnetii opens lines of investigation which should lead to solution of the problem, thereby helping to answer the general question of what is the nature of obligate parasitism. An understanding of the pathobiochemistry of Q fever and of its endotoxicosis rests on elucidating closely interrelated regulatory events. Stimulated hepatic transcription and translation of certain RNA and protein species attend the development of the disease, as do phosphorylation and dephosphorylation of central RNA and protein species. Phosphorylation and dephosphorylation are central regulators in protein synthesis. Whole animal experiments differ from those with cultured cells in important responses. Glycogenolysis, hepatic steatogenesis, and lipase activation are obvious examples of such differences. Stimulation of the production of lymphokines and of hormones is absent in HepG2 cells, and insulin seems to be critical in regulating the phosphorylation-dephosphorylation equilibrium which leads to regulation of protein synthesis. Newly synthesized, so far unidentified proteins may be involved in convalescence, re-establishing the homeostasis of the uninfected state. The model of pathobiochemical regulation in Q fever and endotoxicosis may be applicable to other febrile infections and endotoxicoses.

The ability of mononuclear cells to coagulate blood in response to Coxiella burnetii.

Endocarditis is a rather frequent complication of Q fever caused by Coxiella burnetii. We examined the ability of phase I (virulent) or phase II (avirulent) C. burnetii to coagulate blood in the presence of human blood mononuclear cells in vitro. After incubation for 4 h, virulent phase I C. burnetii was an effective stimulant for mononuclear cells. Since this interaction is a potent trigger of blood coagulation through the extrinsic pathway, it could be responsible for the local deposition of fibrin on the surface of infected valves and the development of large vegetations in cases of endocarditis complicating Q fever.

Infectivity, transmission and 16S rRNA sequencing of a rickettsia, Coxiella cheraxi sp. nov., from the freshwater crayfish Cherax quadricarinatus.

A rickettsia-like organism isolated from infected, farm-reared Cherax quadricarinatus was cultured in the yolk sac of developing chicken eggs, but could not be cultured in 3 continuous cell lines, bluegill fry (BF-2), fathead minnow (FHM), and Spodoptera frugiperda (Sf-9). The organism was confirmed by fulfilling Koch's postulates as the aetiological agent of mortalities amongst C. quadricarinatus. When C. quadricarinatus was inoculated with the organism, mortality was 100% at 28 degrees C and 80% at an ambient temperature of 24 degrees C. Horizontal transmission with food and via the waterborne route was demonstrated, but mortalities were lower at 30 and 10% respectively over a 4 wk period. The 16S rRNA sequence of 1325 base pairs of the Gram-negative, obligate intracellular organism was 95.6% homologous to Coxiella burnetii. Of 18 species compared to this rickettsia, the next most closely related bacterium was Legionella pneumophila at 86.7%. The suggested classification of this organism is Order Rickettsiales, family Rickettsiaceae, tribe Rickettsieae, within the genus Coxiella. We suggest it should be named Coxiella cheraxi sp. nov.

Phase II to phase I conversion of Coxiella burneti in immunosuppressed mice.

Coxiella burneti strains 48 and Nine Mile, serologically in phase II, changed their antigenic properties and virulence for guinea pigs and mice upon passaging in immunosuppressed mice. Conversion to phase I of these strains in a system lacking antibody response throws doubt on the importance of specific antibodies in phase variation of C. burneti.

Search for correlates of resistance to virulent challenge in mice immunized with Coxiella burnetii.

Mice immunized with live phase I or phase II Coxiella burnetii, with killed phase I or phase II organisms or with trichloroacetic acid (TCAE) or phenol (PE) extracts were resistant to intraperitoneal infection with phase I C. burnetii irrespective of whether or not they displayed phase I antibody response at the time of virulent challenge. Increased phagocytosis of purified phase I organisms by blood leukocytes or peritoneal exudate cells (PEC) was noticed only in mice with phase I agglutinating antibodies in their sera or peritoneal washings. Passive transfer of resistance was made possible only by sera containing phase I agglutinating antibodies. Adoptive transfer of immunity by spleen cells, but not by PEC, was achieved providing that these cells were taken from mice immunized with live phase I C. burnetii.

Effect of Coxiella burnetii on the stimulation of hexose monophosphate shunt and on superoxide anion production in human polymorphonuclear leukocytes.

Killed Coxiella burnetii (C.b.) cells in phase II but not in phase I had a mild stimulatory effect on hexose monophosphate shunt (HMPS) and superoxide anion production by human polymorphonuclear (PMN) leukocytes. Preincubation of C.b. cells of either phase with serum of a leukocyte donor lacking detectable antibodies to C.b. did not affect the studied activities of PMN leukocytes. In contrast, both HMPS stimulation and superoxide production were enhanced by specific opsonization of C.b. cells with rabbit immune sera containing corresponding phase I and/or phase II antibodies. Stimulatory effect was observed also with lipopolysaccharide-protein-phospholipid (LPS-Pr-Pl) complex but not with lipopolysaccharide-protein (LPS-Pr) complex and with purified lipopolysaccharide (LPS) isolated from phase I C.b. cells. Possible consequences of these findings for explanation of C.b. resistance to intracellular killing by professional phagocytes are discussed.

[Susceptibility of Ornithodoros papillipes (Argasidae) ticks to the rickettsia Coxiella burnetii]. / Vospriimchivost' kleshchei Ornithodoros papillipes (Argasidae) k rikketsii Coxiella burnetii.

A definition of susceptibility of an arthropod to an infectious or invasional agent is given. The susceptibility means a capacity of the arthropod for infection in a result of which the agent either completes a part of its life cycle in the organism of its invertebrate host or reproduces repeatedly in the organs and tissues of its internal medium causing or not causing a noticeable damage. On the model of the tick O. papillipes and Q. fever agent there were shown differences in the resistance of arthropods to Rickettsiae depending on the developmental phase of the invertebrate host. Under otherwise equal conditions the susceptibility of nymphs of O. papillipes to C. burnetii was in nymph II 146 times, in nymph I 280 times and in larvae 50 000 times less than in nymph IV. It has been established that nymphs I, II, and IV engorge blood only 2.5, 14 and 50 times more respectively than larvae. It shows that the success of infection depends to a larger extent on the degree of susceptibility of an arthropod to a corresponding agent than on the quantity of engorged infected blood. The formula of estimation of agent concentration necessary for successful infection of 50 per cent of ticks is given.