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Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes. A comprehensive understanding of Rickettsia characteristics would ultimately improve clinical diagnosis, assessment, and treatment with improved control and prevention of the disease. Functional studies of rickettsial genes are crucial for understanding their role in pathogenesis. This paper describes a procedure for the electroporation of the Rickettsia parkeri strain Tate's Hell with the shuttle vector pRAM18dSFA and the selection of transformed R. parkeri in tick cell culture with antibiotics (spectinomycin and streptomycin). A method is also described for the localization of transformed R. parkeri in tick cells using confocal immunofluorescence microscopy, a useful technique for checking transformation in vector cell lines. Similar approaches are also suitable for the transformation of other rickettsiae.

Since its recognition in 1994 as the causative agent of human flea-borne spotted fever, Rickettsia felis, has been detected worldwide in over 40 different arthropod species. The cat flea, Ctenocephalides felis, is a well-described biological vector of R. felis. Unique to insect-borne rickettsiae, R. felis can employ multiple routes of infection including inoculation via salivary secretions and potentially infectious flea feces into the skin of vertebrate hosts. Yet, little is known of the molecular interactions governing flea infection and subsequent transmission of R. felis. While the obligate intracellular nature of rickettsiae has hampered the function of large-scale mutagenesis strategies, studies have shown the efficiency of mariner-based transposon systems in Rickettsiales. Thus, this study aimed to assess R. felis genetic mutants in a flea transmission model to elucidate genes involved in vector infection. A Himar1 transposase was used to generate R. felis transformants, in which subsequent genome sequencing revealed a transposon insertion near the 3’ end of sca1. Alterations in sca1 expression resulted in unique infection phenotypes. While the R. felis sca1::tn mutant portrayed enhanced growth kinetics compared to R. felis wild-type during in vitro culture, rickettsial loads were significantly reduced during flea infection. As a consequence of decreased rickettsial loads within infected donor fleas, R. felis sca1::tn exhibited limited transmission potential. Thus, the use of a biologically relevant model provides evidence of a defective phenotype associated with R. felis sca1::tn during flea infection.

The genus Rickettsia encompasses a diverse group of obligate intracellular bacteria that are highly virulent disease agents of mankind as well as symbionts of arthropods. Native plasmids of Rickettsia amblyommatis (AaR/SC) have been used as models to construct shuttle vectors for genetic manipulation of several Rickettsia species. Here we report on the isolation of the complete plasmid (pRM658B) from Rickettsia monacensis (IrR/Munich) mutant Rmona658B and the construction of shuttle vectors based on pRM. To identify regions essential for replication, we made vectors containing the dnaA and parA genes of pRM with varying portions of the region surrounding these genes and a selection-reporter cassette conferring resistance to spectinomycin and expression of green fluorescent protein. Rickettsia amblyommatis (AaR/SC), R. monacensis (IrR/Munich), Rickettsia bellii (RML 369-C), Rickettsia parkeri (Tate’s Hell), and Rickettsia montanensis (M5/6) were successfully transformed with shuttle vectors containing pRM parA and dnaA. PCR assays targeting pRM regions not included in the vectors revealed that native pRM was retained in R. monacensis transformants. Determination of native pRM copy number using a plasmid-encoded gene (RM_p5) in comparison to chromosomal encoded gltA indicated reduced copy numbers in R. monacensis transformants. In transformed R. monacensis, native pRM and shuttle vectors with homologous parA and dnaA formed native plasmid-shuttle vector complexes. These studies provide insight on the maintenance of plasmids and shuttle vectors in rickettsiae.IMPORTANCEThis paper describes a new series of plasmid shuttle vectors for the transformation of rickettsiae.Shuttle vectors based on parA and dnaA sequences of the plasmid pRM can be used to transform diverse rickettsia as well as its native host Rickettsia monacensis.Our results provide insight into the maintenance of rickettsial-based shuttle vectors in rickettsiae.

The blacklegged tick, Ixodes scapularis, a species of significant importance to human and animal health, harbors an endosymbiont Rickettsia buchneri sensu stricto. The symbiont is largely restricted to the ovaries, but all life stages can harbor various quantities or lack R. buchneri entirely. The endosymbiont is cultivable in cell lines isolated from embryos of Ixodes ticks. Rickettsia buchneri most readily grows and is maintained in the cell line IRE11 from the European tick, Ixodes ricinus. The line was characterized by light and electron microscopy and used to analyze the growth dynamics of wildtype and GFPuv-expressing R. buchneri. qPCR indicated that the genome copy doubling time in IRE11 was &gt, 7 days. Measurements of fluorescence using a plate reader indicated that the amount of green fluorescent protein doubled every 11 days. Two 23S rRNA probes were tested via RNA FISH on rickettsiae grown in vitro and adapted to evaluate the tissue tropism of R. buchneri in field-collected female I. scapularis. We observed strong positive signals of R. buchneri in the ovaries and surrounding the nucleus of the developing oocytes. Tissue tropism in I. scapularis and in vitro growth dynamics strengthen the contemporary understanding of R. buchneri as a transovarially transmitted, non-pathogenic endosymbiont.

Anaplasma phagocytophilum (Ap), agent of human anaplasmosis, is an intracellular bacterium that causes the second most common tick-borne illness in North America. To address the lack of a genetic system for these pathogens, we used random Himar1 transposon mutagenesis to generate a library of Ap mutants capable of replicating in human promyelocytes (HL-60 cells). Illumina sequencing identified 1195 non-randomly distributed insertions. As the density of mutants was non-saturating, genes without insertions were either essential for Ap, or spared randomly. To resolve this question, we applied a biostatistical method for prediction of essential genes. Since the chances that a transposon was inserted into genomic TA dinucleotide sites should be the same for all loci, we used a Markov chain Monte Carlo model to estimate the probability that a non-mutated gene was essential for Ap. Predicted essential genes included those coding for structural ribosomal proteins, enzymes involved in metabolism, components of the type IV secretion system, antioxidant defense molecules and hypothetical proteins. We have used an in silico post-genomic approach to predict genes with high probability of being essential for replication of Ap in HL-60 cells. These results will help target genes to investigate their role in the pathogenesis of human anaplasmosis.

Although rickettsiae can cause life-threatening infections in humans worldwide, no licensed vaccine is currently available. To evaluate the suitability of live-attenuated vaccine candidates against rickettsioses, we generated a Rickettsia parkeri mutant RPATATE_0245::pLoxHimar (named 3A2) by insertion of a modified pLoxHimar transposon into the gene encoding a phage integrase protein. For visualization and selection, R. parkeri 3A2 expressed mCherry fluorescence and resistance to spectinomycin. Compared to the parent wild type (WT) R. parkeri, the virulence of R. parkeri 3A2 was significantly attenuated as demonstrated by significantly smaller size of plaque, failure to grow in human macrophage-like cells, rapid elimination of Rickettsia and ameliorated histopathological changes in tissues in intravenously infected mice. A single dose intradermal (i.d.) immunization of R. parkeri 3A2 conferred complete protection against both fatal R. parkeri and R. conorii rickettsioses in mice, in association with a robust and durable rickettsiae-specific IgG antibody response. In summary, the disruption of RPATATE_0245 in R. parkeri resulted in a mutant with a significantly attenuated phenotype, potent immunogenicity and protective efficacy against two spotted fever group rickettsioses. Overall, this proof-of-concept study highlights the potential of R. parkeri mutants as a live-attenuated and multivalent vaccine platform in response to emergence of life-threatening spotted fever rickettsioses.

Mutational analysis is an efficient approach to identifying microbial gene function. Until recently, lack of an effective tool for Anaplasmataceae yielding reproducible results has created an obstacle to functional genomics, because surrogate systems, e.g., ectopic gene expression and analysis in E. coli, may not provide accurate answers. We chose to focus on a method for high-throughput generation of mutants via random mutagenesis as opposed to targeted gene inactivation. In our search for a suitable mutagenesis tool, we considered attributes of the Himar1 transposase system, i.e., random insertion into AT dinucleotide sites, which are abundant in Anaplasmataceae, and lack of requirement for specific host factors. We chose the Anaplasma marginale tr promoter, and the clinically irrelevant antibiotic spectinomycin for selection, and in addition successfully implemented non-antibiotic selection using an herbicide resistance gene. These constructs function reasonably well in Anaplasma phagocytophilum harvested from human promyelocyte HL-60 cells or Ixodes scapularis tick cells. We describe protocols developed in our laboratory, and discuss what likely makes them successful. What makes Anaplasmataceae electroporation competent is unknown and manipulating electroporation conditions has not improved mutational efficiency. A concerted effort is needed to resolve remaining problems that are inherent to the obligate intracellular bacteria. Finally, using this approach, we describe the discovery and characterization of a putative secreted effector necessary for Ap survival in HL-60 cells.

Apoptosis is an innate immune response induced by infection in eukaryotes that contributes significantly to protection from pathogens. However, little is known about the role of apoptosis in the interactions of arthropod vectors with the rickettsiae that they transmit. Rickettsia spp. are vector-borne obligately intracellular bacteria and display different degrees of virulence in their eukaryotic hosts. In this study, we found that infection with Rickettsia parkeri (Rp) activated the apoptosis pathway in an Amblyomma americanum tick cell line (AAE2), as evidenced by the loss of phospholipid membrane asymmetry and DNA fragmentations. Additionally, infection with Rp also led to apoptosis activation in cell lines of different tick species. Interestingly, suppressing apoptosis decreased Rp infection and replication, while the activation of apoptosis increased Rp accumulation at the early stage of infection. Moreover, mitochondrion-dependent apoptosis was essential for Rp infection and replication in vector cells, and apoptosis induction required intracellular rickettsia replication. We further showed that Rp utilizes two different survival strategies to modulate apoptosis in the arthropod vectors and mammalian host cells. There was no direct correlation between apoptosis activation in vector cells and rickettsial pathogenicity. These novel findings indicate a possible mechanism whereby apoptosis facilitates infection and replication of a Rickettsia sp. in an arthropod vector. These results contribute to our understanding of how the vector's responses to pathogen infection affect pathogen replication and therefore transmission. IMPORTANCE Rickettsioses, infections caused by the genus Rickettsia, are among the oldest known infectious diseases. Ticks are essential arthropod vectors for rickettsiae, and knowledge about the interactions between ticks, their hosts, and pathogens is fundamental for identifying drivers of tick-borne rickettsioses. Despite the rapid development in apoptosis research with rickettsiae, little is known regarding the role of apoptosis in the interactions between Rickettsia spp., vertebrate hosts, and arthropod vectors. Here, we demonstrated that mitochondrion-dependent apoptosis is essential for rickettsial infection and replication in vector cells and that apoptosis induction requires intracellular rickettsial replication. However, rickettsial pathogenicity is not linked with apoptosis activation in tick cells. Our findings improve understanding of the apoptosis mechanism in arthropods exploited by rickettsiae and also the potential to discover specific targets for new vaccines and drugs to prevent or treat rickettsial infections.

Lyme disease, the most common vector-borne illness in North America, is caused by the spirochete Borrelia burgdorferi. Infection begins in the skin following a tick bite and can spread to the hearts, joints, nervous system, and other organs. Diverse host responses influence the level of B. burgdorferi infection in mice and humans. Using a systems biology approach, we examined potential molecular interactions between human extracellular and secreted proteins and B. burgdorferi. A yeast display library expressing 1031 human extracellular proteins was probed against 36 isolates of B. burgdorferi sensu lato. We found that human Peptidoglycan Recognition Protein 1 (PGLYRP1) interacted with the vast majority of B. burgdorferi isolates. In subsequent experiments, we demonstrated that recombinant PGLYRP1 interacts with purified B. burgdorferi peptidoglycan and exhibits borreliacidal activity, suggesting that vertebrate hosts may use PGLYRP1 to identify B. burgdorferi. We examined B. burgdorferi infection in mice lacking PGLYRP1 and observed an increased spirochete burden in the heart and joints, along with splenomegaly. Mice lacking PGLYRP1 also showed signs of immune dysregulation, including lower serum IgG levels and higher levels of IFNγ, CXCL9, and CXCL10.Taken together, our findings suggest that PGLYRP1 plays a role in the host’s response to B. burgdorferi and further demonstrate the utility of expansive yeast display screening in capturing biologically relevant interactions between spirochetes and their hosts., Author summary Lyme disease is the most common vector-borne illness in North America and is caused by the spirochete Borrelia burgdorferi. The disease starts with a tick bite that leads to a skin rash and inflammation in other organs of the body, such as hearts and joints. B. burgdorferi uses many strategies to evade detection and persist in the human host. It is important to have efficient methods to be able to identify the various components of the immune system that interact with B. burgdorferi to better understand the disease process, but few currently exist. In this study, we used a novel yeast display screening assay of over 1,000 human immune proteins probed against several isolates of Borrelia to uncover biologically relevant interactions for the Lyme disease pathogen. We identified Peptidoglycan Recognition Protein 1 (PGLYRP1), an innate immune protein important in defense against bacteria, as a major candidate from this screen. We validated the interaction of PGLYRP1 with Borrelia and were able to use PGLYRP1-deficient mice as a model to understand the role of this protein in Lyme disease pathogenesis. Our study demonstrates the potential implications of yeast screens in uncovering important host-pathogen interactions.

Ehrlichia chaffeensis, a tick-transmitted obligate intracellular rickettsial agent, causes human monocytic ehrlichiosis. In recent reports, we described substantial advances in developing random and targeted gene disruption methods to investigate the functions of E. chaffeensis genes. We reported earlier that the Himar1 transposon-based random mutagenesis is a valuable tool in defining E. chaffeensis genes critical for its persistent growth in vivo in reservoir and incidental hosts. The method also aided in extending studies focused on vaccine development and immunity. Here, we describe the generation and mapping of 55 new mutations. To define the critical nature of the bacterial genes, infection experiments were carried out in the canine host with pools of mutant organisms. Infection evaluation in the physiologically relevant host by molecular assays and by xenodiagnoses allowed the identification of many proteins critical for the pathogen’s persistent in vivo growth. Genes encoding proteins involved in biotin biosynthesis, protein synthesis and fatty acid biosynthesis, DNA repair, electron transfer, and a component of a multidrug resistance (MDR) efflux pump were concluded to be essential for the pathogen’s in vivo growth. Three known immunodominant membrane proteins, i.e., two 28-kDa outer membrane proteins (P28/OMP) and a 120-kDa surface protein, were also recognized as necessary for the pathogen’s obligate intracellular life cycle. The discovery of many E. chaffeensis proteins crucial for its continuous in vivo growth will serve as a major resource for investigations aimed at defining pathogenesis and developing novel therapeutics for this and related pathogens of the rickettsial family Anaplasmataceae.

Rickettsia buchneri is the principal symbiotic bacterium of the medically significant tick Ixodes scapularis. This species has been detected primarily in the ovaries of adult female ticks and is vertically transmitted, but its tissue tropism in other life stages and function with regard to tick physiology is unknown. In order to determine the function of R. buchneri, it may be necessary to produce ticks free from this symbiont. We quantified the growth dynamics of R. buchneri naturally occurring in I. scapularis ticks throughout their life cycle and compared it with bacterial growth in ticks in which symbiont numbers were experimentally reduced or eliminated. To eliminate the bacteria, we exposed ticks to antibiotics through injection and artificial membrane feeding. Both injection and membrane feeding of the antibiotic ciprofloxacin were effective at eliminating R. buchneri from most offspring of exposed females. Because of its effectiveness and ease of use, we have determined that injection of ciprofloxacin into engorged female ticks is an efficient means of clearing R. buchneri from the majority of progeny. IMPORTANCE This paper describes the growth of symbiotic Rickettsia buchneri within Ixodes scapularis through the life cycle of the tick and provides methods to eliminate R. buchneri from I. scapularis ticks.

In Norway, the tick-transmitted bacterium Anaplasma phagocytophilum is estimated to cause tick-borne fever (TBF) in 300 000 lambs on pastures each year, resulting in economic and animal welfare consequences. Today, prophylactic measures mainly involve the use of acaricides, but a vaccine has been requested by farmers and veterinarians for decades. Several attempts have been made to produce a vaccine against A. phagocytophilum including antigenic surface proteins, inactivated whole cell vaccines and challenge followed by treatment. In the current study, a virulent wild type strain of A. phagocytophilum named Ap.Norvar1 (16S rRNA sequence partial identical to sequence in GenBank acc.no M73220) was subject to genetic transformation with a Himar1-transposon, which resulted in three bacterial mutants, capable of propagation in a tick cell line (ISE6). In order to test the immunogenicity and pathogenicity of the live, mutated bacteria, these were clinically tested in an inoculation- and challenge study in sheep. One group was inoculated with the Ap.Norvar1 as an infection control. After inoculation, the sheep inoculated with mutated bacteria and the Ap.Norvar1 developed typical clinical signs of infection and humoral immune response. After challenge with Ap.Norvar1, 28 days later all groups inoculated with mutated bacteria showed clinical signs of tick-borne fever and bacteremia while the group initially inoculated with the Ap.Norvar1, showed protection against clinical disease. The current study shows a weak, but partial protection against infection in animals inoculated with mutated bacteria, while animals that received Ap.Norvar1 both for inoculation and challenge, responded with homologues protection. Funding Agencies|Norwegian research funding for agriculture and the food industry (Matfondavtalen) [244173/E50]; Norwegian Association of Sheep and Goat Farmers (NSG); Animalia - the Norwegian Meat and Poultry Research Center; ScandTick Innovation (Eu-Interreg OKS IVA); NorthTick (the EU-Interreg North Sea Region program)

Knowledge of the T4SS is derived from model systems, such as Agrobacterium tumefaciens. The structure of the T4SS in Rickettsiales differs from the classical arrangement. These differences include missing and duplicated components with structural alterations. Particularly, two sequenced virB6-4 genes encode unusual C-terminal structural extensions resulting in proteins of 4,322 (GenBank accession number AGR79286.1) and 9,935 (GenBank accession number ANC34101.1) amino acids. To understand how the T4SS is used in A. phagocytophilum, we describe the expression of the virB6 paralogs and explore their role as the bacteria replicate within its host cell. Conclusions about the importance of these paralogs for colonization of human and tick cells are supported by the deficient phenotype of an A. phagocytophilum mutant isolated from a sequence-defined transposon insertion library., Many pathogenic bacteria translocate virulence factors into their eukaryotic hosts by means of type IV secretion systems (T4SS) spanning the inner and outer membranes. Genes encoding components of these systems have been identified within the order Rickettsiales based upon their sequence similarities to other prototypical systems. Anaplasma phagocytophilum strains are obligate intracellular, tick-borne bacteria that are members of this order. The organization of these components at the genomic level was determined in several Anaplasma phagocytophilum strains, showing overall conservation, with the exceptions of the virB2 and virB6 genes. The virB6 loci are characterized by the presence of four virB6 copies (virB6-1 through virB6-4) arranged in tandem within a gene cluster known as the sodB-virB operon. Interestingly, the virB6-4 gene varies significantly in length among different strains due to extensive tandem repeats at the 3′ end. To gain an understanding of how these enigmatic virB6 genes function in A. phagocytophilum, we investigated their expression in infected human and tick cells. Our results show that these genes are expressed by A. phagocytophilum replicating in both cell types and that VirB6-3 and VirB6-4 proteins are surface exposed. Analysis of an A. phagocytophilum mutant carrying the Himar1 transposon within the virB6-4 gene demonstrated that the insertion not only disrupted its expression but also exerted a polar effect on the sodB-virB operon. Moreover, the altered expression of genes within this operon was associated with the attenuated in vitro growth of A. phagocytophilum in human and tick cells, indicating the importance of these genes in the physiology of this obligate intracellular bacterium in such different environments. IMPORTANCE Knowledge of the T4SS is derived from model systems, such as Agrobacterium tumefaciens. The structure of the T4SS in Rickettsiales differs from the classical arrangement. These differences include missing and duplicated components with structural alterations. Particularly, two sequenced virB6-4 genes encode unusual C-terminal structural extensions resulting in proteins of 4,322 (GenBank accession number AGR79286.1) and 9,935 (GenBank accession number ANC34101.1) amino acids. To understand how the T4SS is used in A. phagocytophilum, we describe the expression of the virB6 paralogs and explore their role as the bacteria replicate within its host cell. Conclusions about the importance of these paralogs for colonization of human and tick cells are supported by the deficient phenotype of an A. phagocytophilum mutant isolated from a sequence-defined transposon insertion library.

The incidence of human diseases caused by tick-borne pathogens is increasing but little is known about the molecular interactions between the agents and their vectors and hosts. Anaplasma phagocytophilum (Ap) is an obligate intracellular, tick-borne bacterium that causes granulocytic anaplasmosis in humans, dogs, sheep, and horses. In mammals, neutrophil granulocytes are a primary target of infection, and in ticks, Ap has been found in gut and salivary gland cells. To identify bacterial genes that enable Ap to invade and proliferate in human and tick cells, labeled mRNA from Ap bound to or replicating within human and tick cells (lines HL-60 and ISE6), and replicating in primary human granulocytes ex vivo, was hybridized to a custom tiling microarray containing probes representing the entire Ap genome. Probe signal values plotted over a map of the Ap genome revealed antisense transcripts and unannotated genes. Comparisons of transcript levels from each annotated gene between test conditions (e.g., Ap replicating in HL-60 vs. ISE6) identified those that were differentially transcribed, thereby highlighting genes associated with each condition. Bacteria replicating in HL-60 cells upregulated 122 genes compared to those in ISE6, including numerous p44 paralogs, five HGE-14 paralogs, and 32 hypothetical protein genes, of which 47% were predicted to be secreted or localized to the membrane. By comparison, 60% of genes upregulated in ISE6 encoded hypothetical proteins, 60% of which were predicted to be secreted or membrane associated. In granulocytes, Ap upregulated 120 genes compared to HL-60, 33% of them hypothetical and 43% of those predicted to encode secreted or membrane associated proteins. HL-60-grown bacteria binding to HL-60 cells barely responded transcriptionally, while ISE6-grown bacteria binding to ISE6 cells upregulated 48 genes. HL-60-grown bacteria, when incubated with ISE6 cells, upregulated the same genes that were upregulated by ISE6-grown bacteria exposed to uninfected ISE6. Hypothetical genes (constituting about 29% of Ap genes) played a disproportionate role in most infection scenarios, and particular sets of them were consistently upregulated in bacteria binding/entering both ISE6 and HL-60 cells. This suggested that the encoded proteins played central roles in establishing infection in ticks and humans.

BACKGROUNDHaematobia spp., horn flies (HF) and buffalo flies (BF), are economically important ectoparasites of dairy and beef cattle. Control of these flies relies mainly on the treatment of cattle with chemical insecticides. However, the development of resistance to commonly used compounds is compromising the effectiveness of these treatments and alternative methods of control are required. Wolbachia are maternally transmitted endosymbiotic bacteria of arthropods that cause various reproductive distortions and fitness effects, making them a potential candidate for use in the biological control of pests.RESULTSHere we report the successful establishment of a continuous HF cell line (HIE-18) from embryonic cells and its stable transinfection with Wolbachia strain wAlbB native to mosquitoes, and wMel and wMelPop native to Drosophila melanogaster. The established HIE-18 cells are typically round and diploid with ten chromosomes (2n = 10) or tetraploid with 20 chromosomes (4n=20) having a doubling time of 67.2 hours. Wolbachia density decreased significantly in the HIE-18 cells in the first 48 hours of infection, possibly due to overexpression of antimicrobial peptides through the Imd immune signalling pathway. However, density recovered after this time and stably Wolbachia-infected HIE-18 cell lines have now all been subcultured more than 50 times as persistently infected lines.CONCLUSIONThe amenability of HF to infection with different strains of Wolbachia suggests the potential for use of Wolbachia in novel approaches for the control of Haematobia spp. Further, the availability of the HIE-18 cell line will provide an important resource for the study of genetics, host-parasite interactions and chemical resistance in Haematobia populations.

Haematobia spp., horn flies (HF) and buffalo flies (BF), are economically important ectoparasites of dairy and beef cattle. Control of these flies relies mainly on the treatment of cattle with chemical insecticides. However, the development of resistance to commonly used compounds is compromising the effectiveness of these treatments and alternative methods of control are required. Wolbachia are maternally transmitted endosymbiotic bacteria of arthropods that cause various reproductive distortions and fitness effects, making them a potential candidate for use in the biological control of pests. The first step towards this is the establishment and adaptation of xenobiotic infections of Wolbachia in target host cell lines. Here we report the successful establishment of a continuous HF cell line (HIE-18) from embryonic cells and its stable transinfection with Wolbachia strains wAlbB native to mosquitoes, and wMel and wMelPop native to Drosophila melanogaster. The HIE-18 cells were typically round and diploid with ten chromosomes (2n = 10) or tetraploid with 20 chromosomes (4n = 20) with a doubling time of 67.2 h. Wolbachia density decreased significantly in the HIE-18 cells in the first 48 h of infection, possibly due to overexpression of antimicrobial peptides through the Imd immune signalling pathway. However, density recovered after this time and HIE-18 cell lines stably infected with the three strains of Wolbachia have now each been subcultured more than 50 times as persistently infected lines. The amenability of HF cells to infection with different strains of Wolbachia and the establishment of stable sustaining infections suggests the potential for use of Wolbachia in novel approaches for the control of Haematobia spp. Further, the availability of the HIE-18 cell line will provide an important resource for the study of genetics, host-parasite interactions and chemical resistance in Haematobia populations. This article is protected by copyright. All rights reserved.

Infections with arthropod-borne pathogens are an increasing threat world-wide that requires heightened vigilance from veterinary and medical practitioners, especially when they involve new or unusual organisms. A dog was presented to a local veterinary clinic in Germany with malaise, pale mucous membranes and stiff joints. Clinical assessment revealed pyrexia, leukopenia and thrombocytopenia. On suspicion of a tick-borne infection, blood samples were examined for clinical and biochemical parameters and subjected to a Anaplasma phagocytophilum -, Borrelia spp.- and Ehrlichia canis -specific real-time PCR. Additionally, a sample of the pre-therapeutic buffy coat was co-cultured with the Ixodes scapularis cell-line ISE6 for 20 days. Only the PCR specific for A. phagocytophilum DNA yielded a positive result, and furthermore, Anaplasma morulae were visible in granulocytes and tick cells. After co-culturing, extracellular trypomastigote and epimastigote stages of Trypanosoma sp. with an average length of 29.7 μm were observed, featuring a pointed posterior end. Sequence analysis of a 2080 bp fragment of the 18S rRNA gene showed 99% identity to the 18S rRNA gene of Trypanosoma pestanai , previously described from a European badger ( Meles meles ) in France. The dog's condition improved rapidly in response to doxycycline treatment for three weeks. The clinical status normalized and clinical blood parameters were found to be within the reference ranges. To our knowledge this is the first description of T. pestanai infection in a dog, the first detection of T. pestanai in Germany and the first documented co-infection with these two pathogens. Co-infections with unusual opportunistic vector-borne pathogens should be considered, if acute canine granulocytic anaplasmosis is evident.

We have previously described a novel taxon of the genus Ehrlichia (type strain WisconsinT), closely related to Ehrlichia muris, that causes human ehrlichiosis among patients with exposures to ticks in the upper midwestern USA. DNA from this bacterium was also detected in Ixodes scapularis and Peromyscus leucopus collected in Minnesota and Wisconsin. To determine the relationship between the E. muris-like agent (EMLA) and other species of the genus Ehrlichia phenotypic, genotypic and epidemiologic comparisons were undertaken, including sequence analysis of eight gene loci (3906 nucleotides) for 39 EMLA DNA samples and the type strain of E. muris AS145T. Three loci were also sequenced from DNA of nine strains of E. muris from mouse spleens from Japan. All sequences from E. muris were distinct from homologous EMLA sequences, but differences between them were less than those observed among other species of the genus Ehrlichia. Phenotypic comparison of EMLA and E. muris revealed similar culture and electron microscopic characteristics, but important differences were noted in their geographic distribution, ecological associations and behavior in mouse models of infection. Based on these comparisons, we propose that type strain WisconsinT represents a novel subspecies, Ehrlichia murissubsp. eauclairensis,subsp. nov. This strain is available through the Centers for Disease Control and Prevention Rickettsial Isolate Reference Collection (CRIRC EMU002T) and through the Collection de Souches de l’Unité des Rickettsies (CSURP2883 T). The subspecies Ehrlichia murissubsp. muris subsp. nov. is automatically created and the type strain AS145T is also available through the same collections (CRIRC EMU001T, CSUR E2T). Included is an emended description of E. muris.

Human diseases caused by bacteria in the genus Rickettsia , commonly referred to as typhus or spotted fevers, are among the oldest and most severe scourges of humankind. All of the diseases’ agents utilize blood-sucking arthropods as vectors, which influences disease epidemiology. Epidemic typhus continues to cause outbreaks today in situations of war and unrest (1), and the incidence of Rocky Mountain spotted fever is growing in the Americas (2). Despite an urgent need, broadly protective vaccines are unavailable, although previously used vaccines proved this an achievable goal (3). It has long been known that recovered patients mount protective immune responses, producing antibodies that have been used for diagnosis of rickettsioses through the so-called Weil−Felix reaction since the early 1900s (4). However, until the research by Kim et al. (5), the identity of the genes encoding the molecular machinery responsible for producing the cognate antigen in all species of rickettsiae was unknown. The history of the Western world reflects the power of rickettsial disease agents. Epidemic typhus, which may kill over 30% of infected people, has shaped history profoundly at least since medieval times (6), when hapless inhabitants of cities beleaguered by enemy armies were forced to live in crowded quarters and under unsanitary conditions. This created favorable conditions for human body lice, the vectors for the causative agent, Rickettsia prowazekii (7). Soldiers … [↵][1]1Email: munde001{at}umn.edu. [1]: #xref-corresp-1-1

Ehrlichia muris subsp. eauclairensis is recognized as the etiological agent of human ehrlichiosis in Minnesota and Wisconsin. We describe the culture isolation of this organism from a field-collected tick and detail its relationship to other species of Ehrlichia. The isolate could be grown in a variety of cultured cell lines and was effectively transmitted between Ixodes scapularis ticks and rodents, with PCR and microscopy demonstrating a broad pattern of dissemination in arthropod and mammalian tissues. Conversely, Amblyomma americanum ticks were not susceptible to infection by the Ehrlichia. Histologic sections further revealed that the wild-type isolate was highly virulent for mice and hamsters, causing severe systemic disease that was frequently lethal. A Himar1 transposase system was used to create mCherry- and mKate-expressing EmCRT mutants, which retained the ability to infect rodents and ticks. IMPORTANCE Ehrlichioses are zoonotic diseases caused by intracellular bacteria that are transmitted by ixodid ticks. Here we report the culture isolation of bacteria which are closely related to, or the same as the Ehrlichia muris subsp. eauclairensis, a recently recognized human pathogen. EmCRT, obtained from a tick removed from deer at Camp Ripley, MN, is the second isolate of this subspecies described and is distinctive in that it was cultured directly from a field-collected tick. The isolate’s cellular tropism, pathogenic changes caused in rodent tissues, and tick transmission to and from rodents are detailed in this study. We also describe the genetic mutants created from the EmCRT isolate, which are valuable tools for the further study of this intracellular pathogen.

Microsporidia are important natural regulators of many insect populations and as such are suitable microbial agents for the biological control of pest insects. Among the best-characterized microsporidia are those that infect the European corn borer, Ostrinia nubilalis. This chapter presents the analysis on Nosema pyrausta and Vairimorpha necatrix, as well as the recently described Nosema fur-nacalis. It examines the criteria in light of the need to recognize or develop more effective strains of microsporidia for corn borer control. The chapter reviews the advances in the identification of microsporidia that may facilitate the detection of introduced microsporidia and their delineation from indigenous species. It aims to evaluate what is known about microsporidian strains that offer different characteristics important to their use in biological control and the prospects for their manipulation via strain selection or genetic manipulation. Genetic studies and specific identification based on cytogenetic features are difficult because of the small size of microsporidia and their intracellular inhabitation.

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Ticks are obligate hematophagous arthropods and must tolerate starvation during off-host periods. Macroautophagy (hereafter autophagy) is a well-conserved self-eating mechanism of cell survival and is essential for recycling cellular contents during periods of starvation, stress, and injury in organisms. Although the genome sequence of Ixodes scapularis (Say) is available, the characteristics and functions of autophagy-related gene families remain largely unknown. To advance our understanding of autophagy in I. scapularis, we used comprehensive genomic approaches to identify Atg genes. Homologues of 14 Atg genes were identified, and their protein motif compositions were predicted. Phylogenetic analysis indicated that ATGs in I. scapularis were evolutionarily closely related to their homologues in Haemaphysalis longicornis and Rhipicephalus microplus ticks. Expression patterns of Atg genes differed across tick developmental stages. Immunofluorescence results by monodansylcadaverine (MDC) staining indicated that autophagy was activated after amino acid starvation treatments in I. scapularis embryo-derived cell lines ISE6 and IDE8. Subsequently, the expression of key Atg genes involved in autophagy pathway in both cell lines were examined. In ISE6 cells, the expression levels of three Atg genes (Atg4B, Atg6 and Atg8A) increased significantly after amino acid starvation; similarly, four Atg genes (Atg4A, Atg4B, Atg6 and Atg8B) were upregulated in IDE8 cells in response to starvation. In parallel, the MDC and lysotracker staining results indicated that autophagy was triggered after amino acid starvation treatments in R. microplus embryo-derived cell line BME26. Our observations showed that Atg family genes are highly conserved in ticks and function in autophagy pathway induced by amino acid starvation. These results also provide valuable insight for further autophagy-related research as a new strategy for blocking the transmission of tick-borne pathogens.

Anaplasma marginale is the causative agent of anaplasmosis in cattle. Transposon mutagenesis of this pathogen using the Himar1 system resulted in the isolation of an omp10 operon insertional mutant referred to as the omp10 :: himar1 mutant. The work presented here evaluated if this mutant had morphological and/or growth rate defects compared to wild-type A. marginale . Results showed that the morphology, developmental cycle, and growth in tick and mammalian cell cultures are similar for the mutant and the wild type. Tick transmission experiments established that tick infection levels with the mutant were similar to those with wild-type A. marginale and that infected ticks successfully infected cattle. However, this mutant exhibited reduced infectivity and growth in cattle. The possibility of transforming A. marginale by transposon mutagenesis coupled with in vitro and in vivo assessment of altered phenotypes can aid in the identification of genes associated with virulence. The isolation of deliberately attenuated organisms that can be evaluated in their natural biological system is an important advance for the rational design of vaccines against this species.

We obtained a rickettsial isolate from the ovaries of the blacklegged tick, Ixodes scapularis. The isolate (ISO7T) was grown in the Ixodes ricinus embryonic cell line IRE11. We characterized the isolate by transmission electron microscopy and gene sequencing. Phylogenetic analysis of 11 housekeeping genes demonstrated that the isolate fulfils the criteria to be classified as a representative of a novel rickettsial species closely related to ‘Rickettsia monacensis’. These rickettsiae form a clade separate from other species of rickettsiae. Gene sequences indicated that several genes important in rickettsial motility, invasiveness and temperature adaptation were mutated (e.g. sca2, rickA, hsp22, pldA and htrA). We propose the name Rickettsia buchneri sp. nov. for this bacterium that infects the ovaries of the tick I. scapularis to acknowledge the pioneering contributions of Professor Paul Buchner (1886–1978) to research on bacterial symbionts. The type strain of R. buchneri sp. nov. is strain ISO-7T ( = DSM 29016T = ATCC VR-1814T).

Anaplasma phagocytophilum (Ap) is a tick-borne pathogen, which can cause granulocytic anaplasmosis in humans and animals. In vivo this obligate intracellular pathogen is primarily located in circulating mature granulocytes, but it also infects endothelial cells. In order to study the interaction between Ap-infected endothelial cells and human granulocytes under conditions similar to those found naturally in the infected host, an in vitro model that mimics physiological flow conditions in the microvasculature was established. Cell-to-cell interactions were then visualized by microscopy, which showed that granulocytes adhered strongly to Ap-infected endothelial cells at a shear stress of 0.5 dyne/cm(2). In addition, Ap-transmission assays under flow conditions showed that the bacteria transferred from infected endothelial cells to circulating granulocytes and were able to establish infection in constantly moving granulocytes. Cell surface analysis showed that Ap induced up-regulation of the cell adhesion molecules ICAM-1 and VCAM-1 on infected endothelial cells in a dose-dependent manner. Furthermore, IL-8 secretion by endothelial cells indicated that the presence of Ap induced a pro-inflammatory response. In summary, the results of this study suggest that endothelial cells of the microvasculature (1) provide an excellent site for Ap dissemination to peripheral blood granulocytes under flow conditions and therefore may play a crucial role in the development of persistent infection, and (2) are stimulated by Ap to express surface molecules and cytokines that may lead to inflammatory responses at the site of the infection.

It is estimated that approximately one billion people are at risk of infection with obligate intracellular bacteria, but little is known about the underlying mechanisms that govern their life cycles. The difficulty in studying Chlamydia spp., Coxiella spp., Rickettsia spp., Anaplasma spp., Ehrlichia spp. and Orientia spp. is, in part, due to their genetic intractability. Recently, genetic tools have been developed; however, optimizing the genomic manipulation of obligate intracellular bacteria remains challenging. In this Review, we describe the progress in, as well as the constraints that hinder, the systematic development of a genetic toolbox for obligate intracellular bacteria. We highlight how the use of genetically manipulated pathogens has facilitated a better understanding of microbial pathogenesis and immunity, and how the engineering of obligate intracellular bacteria could enable the discovery of novel signalling circuits in host-pathogen interactions.

Rickettsia typhi is the causative agent of endemic typhus, a disease with increasing incidence worldwide that can be fatal. Because of its obligate intracellular life style, genetic manipulation of the pathogen is difficult. Nonetheless, in recent years, genetic manipulation tools have been successfully applied to rickettsiae. We describe here for the first time the transformation of R. typhi with the pRAM18dRGA plasmid that originally derives from Rickettsia amblyommatis and encodes the expression of GFPuv (green fluorescent protein with maximal fluorescence when excited by UV light). Transformed R. typhi ( R. typhi GFPuv ) bacteria are viable, replicate with kinetics similar to those of wild-type R. typhi in cell culture, and stably maintain the plasmid and GFPuv expression under antibiotic treatment in vitro and in vivo during infection of mice. CB17 SCID mice infected with R. typhi GFPuv succumb to the infection with kinetics similar to those for animals infected with wild-type R. typhi and develop comparable pathology and bacterial loads in the organs, demonstrating that the plasmid does not influence pathogenicity. In the spleen and liver of infected CB17 SCID mice, the bacteria are detectable by immunofluorescence microscopy in neutrophils and macrophages by histological staining. Finally, we show for the first time that transformed rickettsiae can be used for the detection of CD8 + T cell responses. GFP-specific restimulation of spleen cells from R. typhi GFPuv -infected BALB/c mice elicits gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 2 (IL-2) secretion by CD8 + T cells. Thus, R. typhi GFPuv bacteria are a novel, potent tool to study infection with the pathogen in vitro and in vivo and the immune response to these bacteria.

Ticks transmit a variety of human pathogens, including Borrelia burgdorferi, the etiological agent of Lyme disease. Multiple pathogens that are transmitted simultaneously, termed “coinfections”, are of increasing importance and can affect disease outcome in a host. Arthropod immunity is central to pathogen acquisition and transmission by the tick. Pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) and induce humoral responses through the Toll and Immune Deficiency (IMD) pathways. Comparative analyses between insects and ticks reveal that while the Toll pathway is conserved, the IMD network exhibits a high degree of variability. This indicates that major differences in humoral immunity exist between insects and ticks. While many variables can affect immunity, one of the major forces that shape immune outcomes is the microbiota. In light of this, we discuss how the presence of commensal bacteria, symbionts and/or coinfections can lead to altered immune responses in the tick that impact pathogen persistence and subsequent transmission. By investigating non-insect arthropod immunity, we will not only better comprehend tick biology, but also unravel the intricate effects that pathogen coinfections have on vector competence and tick-borne disease transmission.

Background The Ehrlichia muris-like agent (EMLA) is a newly recognized human pathogen in the North Central United States. Although blacklegged ticks (Ixodes scapularis) have been identified as capable vectors, wild reservoirs have not yet been established for EMLA. As key hosts for I. scapularis, white-footed mice (Peromyscus leucopus) are important reservoirs for various tick-borne pathogens, and potentially, for EMLA. The objective of this study was to evaluate reservoir competence in P. leucopus using a natural vector. Results Mice acquired EMLA infection from feeding ticks and were able to transmit infection to naïve ticks. Transmission between simultaneously feeding tick life stages was also demonstrated. Infections in mice were acute and severe, with systemic dissemination. Limited host survival and clearance of infection among survivors resulted in a narrow interval where EMLA could be acquired by feeding ticks. Conclusions Peromyscus leucopus is a competent reservoir of EMLA and likely to play a role in its enzootic transmission cycle. The duration and severity of EMLA infection in these hosts suggests that tick phenology is a critical factor determining the geographic distribution of EMLA in North America.

As humans and climate change alter the landscape, novel disease risk scenarios emerge. Understanding the complexities of pathogen emergence and subsequent spread as shaped by landscape heterogeneity is crucial to understanding disease emergence, pinpointing high-risk areas, and mitigating emerging disease threats in a dynamic environment. Tick-borne diseases present an important public health concern and incidence of many of these diseases are increasing in the United States. The complex epidemiology of tick-borne diseases includes strong ties with environmental factors that influence host availability, vector abundance, and pathogen transmission. Here, we used 16 years of case data from the Minnesota Department of Health to report spatial and temporal trends in Lyme disease (LD), human anaplasmosis, and babesiosis. We then used a spatial regression framework to evaluate the impact of landscape and climate factors on the spread of LD. Finally, we use the fitted model, and landscape and climate datasets projected under varying climate change scenarios, to predict future changes in tick-borne pathogen risk. Both forested habitat and temperature were important drivers of LD spread in Minnesota. Dramatic changes in future temperature regimes and forest communities predict rising risk of tick-borne disease.

The rickettsial protein RickA activates host cell factors associated with the eukaryotic actin cytoskeleton and is likely involved with rickettsial host cell binding and infection and the actin-based motility of spotted fever group rickettsiae. The rickA gene sequence and protein vary substantially between Rickettsia species, as do observed motility-associated phenotypes. To help elucidate the function of RickA and determine the effects of species-specific RickA variations, we compared extracellular binding, intracellular motility, and intercellular spread phenotypes of three Rickettsia bellii variants. These included two shuttle vector-transformed R. bellii strains and the wild-type isolate from which they were derived, R. bellii RML 369C. Both plasmid shuttle vectors carried spectinomycin resistance and a GFPuv reporter; one contained Rickettsia monacensis -derived rickA , and the other lacked the rickA gene. Rickettsia bellii transformed to express R. monacensis rickA highly overexpressed this transcript in comparison to its native rickA . These rickettsiae also moved at higher velocities and followed a more curved path than the negative-control transformants. A lower proportion of R. monacensis rickA -expressing bacteria ever became motile, however, and they formed smaller plaques.

Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that provokes an acute inflammatory response during mammalian infection. The illness caused by A. phagocytophilum , human granulocytic anaplasmosis, occurs irrespective of pathogen load and results instead from host-derived immunopathology. Thus, characterizing A. phagocytophilum genes that affect the inflammatory process is critical for understanding disease etiology. By using an A. phagocytophilum Himar1 transposon mutant library, we showed that a single transposon insertion into the A. phagocytophilum dihydrolipoamide dehydrogenase 1 gene ( lpda1 [ APH _ 0065 ]) affects inflammation during infection. A. phagocytophilum lacking lpda1 revealed enlargement of the spleen, increased splenic extramedullary hematopoiesis, and altered clinicopathological abnormalities during mammalian colonization. Furthermore, LPDA1-derived immunopathology was independent of neutrophil infection and correlated with enhanced reactive oxygen species from NADPH oxidase and nuclear factor (NF)-κB signaling in macrophages. Taken together, these findings suggest the presence of different signaling pathways in neutrophils and macrophages during A. phagocytophilum invasion and highlight the importance of LPDA1 as an immunopathological molecule.

A reduction in the use of animals in infectious disease research is desirable for animal welfare as well as for simplification and standardization of experiments. An artificial silicone-based membrane-feeding system was adapted for complete engorgement of adult and nymphal Ixodes scapularis Say (Acari: Ixodidae), and for infecting nymphs with pathogenic, tick-borne bacteria. Six wild-type and genetically transformed strains of four species of bacteria were inoculated into sterile bovine blood and fed to ticks. Pathogens were consistently detected in replete nymphs by polymerase chain reaction. Adult ticks that ingested bacteria as nymphs were evaluated for transstadial transmission. Borrelia burgdorferi and Ehrlichia muris -like agent showed high rates of transstadial transmission to adult ticks, whereas Anaplasma phagocytophilum and Rickettsia monacensis demonstrated low rates of transstadial transmission/maintenance. Artificial membrane feeding can be used to routinely maintain nymphal and adult I. scapularis , and infect nymphs with tick-borne pathogens.