Resistance of the Tick Dermacentor variabilis (Acari: Ixodidae) Following Challenge with the Bacterium Escherichia coli (Enterobacteriales: Enterobacteriaceae)

In addition to a soluble response, many invertebrates control bacterial infections by means of phagocytosis or melanotic encapsulation. In some insects, Escherichia coli growth is reported to be inhibited by aggregation/encapsulation. Soluble and phagocytic responses to bacterial challenge have been reported in ticks, but evidence of an aggregation/encapsulation response was reported only for inanimate (araldite) implants. Ticks were challenged by direct inoculation of bacteria into the hemocoel cavity. By plating, no viable E. coli were detected 6 h postinoculation. A direct fluorescence assay (DFA) revealed aggregated bacteria 1 h postinoculation. Furthermore, DFA showed aggregated bacteria at 6, 24, and 48 h postinoculation associated with masses of tissue, presumably of cellular origin, suggesting events similar to those described as nodulation. These findings suggest that encapsulation/nodulation may be an important component of the immune response in ticks

Genome-wide screen for temperature-regulated genes of the obligate intracellular bacterium, Rickettsia typhi

<p>Abstract</p> <p>Background</p> <p>The ability of rickettsiae to survive in multiple eukaryotic host environments provides a good model for studying pathogen-host molecular interactions. <it>Rickettsia typhi</it>, the etiologic agent of murine typhus, is a strictly intracellular gram negative α-proteobacterium, which is transmitted to humans by its arthropod vector, the oriental rat flea, <it>Xenopsylla cheopis</it>. Thus, <it>R. typhi </it>must cycle between mammalian and flea hosts, two drastically different environments. We hypothesize that temperature plays a role in regulating host-specific gene expression, allowing <it>R. typhi </it>to survive in mammalian and arthropod hosts. In this study, we used Affymetrix microarrays to screen for temperature-induced genes upon a temperature shift from 37°C to 25°C, mimicking the two different host temperatures <it>in vitro</it>.</p> <p>Results</p> <p>Temperature-responsive genes belonged to multiple functional categories including among others, transcription, translation, posttranslational modification/protein turnover/chaperones and intracellular trafficking and secretion. A large number of differentially expressed genes are still poorly characterized, and either have no known function or are not in the COG database. The microarray results were validated with quantitative real time RT-PCR.</p> <p>Conclusion</p> <p>This microarray screen identified various genes that were differentially expressed upon a shift in temperature from 37°C to 25°C. Further characterization of the identified genes may provide new insights into the ability of <it>R. typhi </it>to successfully transition between its mammalian and arthropod hosts.</p

Infection and Transovarial Transmission of Rickettsiae in Dermacentor variabilis Acquired by Artificial Feeding

In this study we examined the efficiency of an in vitro feeding technique using glass microcapillaries as a method of establishing rickettsiae-infected lines of ticks. To quantify the volume ingested by ticks during microcapillary feeding, the incorporation of radiolabeled amino acids in tick gut and hemolymph was calculated. Fifteen of 18 ticks consumed between 0.06 μl and 6.77μl. However, ingestion of fluid was not correlated to weight gain during capillary feeding. Uninfected and partially fed laboratory-reared female Dermacentor variabilis ticks were exposed to either Rickettsia montana- or Rickettsia rhipicephali-infected Vero cells via microcapillary tubes, returned to rabbit hosts, and allowed to feed to repletion. All tissues collected from ticks allowed to feed overnight on rickettsiae-infected fluids were found to be infected when examined by IFA. When rickettsiae-infected and uninfected capillary-fed ticks were allowed to feed to repletion and lay eggs, no significant differences in mean engorgement weight or fecundity was observed. When we assessed the efficiency of transovarial transmission of rickettsiae by ticks that imbibed rickettsiae-infected cells by polymerase chain reaction (PCR) and IFA, infection was detected by PCR in the eggs from 85% of the ticks exposed to R. montana and 69% of the ticks exposed to R. rhipicephali. Rickettsial genes were not amplified in samples of the uninfected controls. Examination by IFA of egg samples from females exposed to rickettsiae-infected cells identified rickettsiae in 100% of the samples tested, while the uninfected controls were negative

An Arthropod Defensin Expressed by the Hemocytes of the American Dog Tick, Dermacentor variabilis (Acari: Ixodidae)

Both soluble and cell-mediated components are involved in the innate immune response of arthropods. Injection of Borrelia burgdorferi, the Lyme disease agent, results in the secretion of defensin into the hemolymph of the ixodid tick, Dermacentor variabilis. The presence of the peptide is observed as early as 15 min post-challenge and remains present through 18 h post-challenge. As observed in insects and soft ticks, the transcript for defensin is detected as early as 1 h post-challenge in D. variabilis. RT-PCR resulted in an amplicon of 624 bp with a 225 bp region that translates to a 74 amino acid preprodefensin. The defensin encoding region was amplified, cloned and sequenced from the hemocytes. It appears as though defensin is stored in the granulocytes of the hemolymph and secreted into the hemolymph upon bacterial insult. The role of defensin as a contributing factor in determining vector competency is discussed

Rickettsial Infection in Dermacentor variabilis (Acari : Ixodidae) Inhibits Transovarial Transmission of a Second Rickettsia

This study examined the ability of ticks to maintain multiple species of spotted fever group rickettsiae via transovarial transmission. Using a capillary feeding method, previously established Rickettsia montana- and Rickettsia rhipicephali-infected cohorts of Dermacentor variabilis (Say) were exposed to R. rhipicephali and R. montana, respectively, in two reciprocal challenge experiments. Eggs collected from individual females, for two successive generations, of each cohort were assessed for rickettsial infection by polymerase chain reaction for each challenge experiment. Assessment of the eggs from challenged ticks identified that both R. montana- and R. rhipicephali-infected ticks were refractory to their respective challenge rickettsiae. The prechallenged infection rate for both F1 and F2 generations (100%) of the R. montana-infected cohort was resistant to transovarial transmission of the second rickettsia species, and only R. montana was detected in the eggs of F1 = (50%) and F2 = (74%) challenged females. The R. rhipicephali-infected cohort maintained a lower level of infection (20%) in the population and did not transovarially transmit the challenge species, however, detectable levels of infection were lost after the first generation. Second-generation ticks, no longer infected with R. rhipicephali, became susceptible to infection with R. montana and female ticks (approximate to 4%) were able to transmit R. montana to their progeny. The resistance of the ovaries to co-infection and apparent host-specific nature of infection suggests that rickettsial infection of tick ovaries may alter the molecular expression of the oocytes so as to preclude secondary infection with other rickettsiae

Host Blood Proteins and Peptides in the Midgut of the Tick Dermacentor variabilis Contribute to Bacterial Control

Antimicrobial midgut proteins and peptides that result from blood digestion in feeding American dog ticks Dermacentor variabilis (Say) were identified. Midgut extracts from these ticks showed antimicrobial activity against Micrococcus luteus, regardless of whether they were challenged with peptidoglycan, blood meal components, rabbit blood, Bacillus subtilis, Escherischia coli or Borrelia burgdorferi. However, no peptide band co-migrating with defensin was found in midgut extracts from the challenged ticks. Partial purification of the midgut extracts using C18 Sep Paks and gel electrophoresis showed the presence of 4 distinct bands with rMW 4.1, 5.3, 5.7 and 8.0 kDa identified by tryptic digestion-mass fingerprinting as digestive fragments of rabbit α-, β-, ᵧ-chain hemoglobin, and rabbit ubiquitin. No evidence of varisin, a defensin previously identified in the hemolymph of D. variabilis, was found in the tryptic digest, although varisin was found in a hemocyte lysate using the same methods. However, varisin transcript was detected in midgut cell lysates. Also present in all midgut samples was a cluster of 3 overlapping bands with rMW 13.0, 14.1 and 14.7 kDa which were identified by tryptic-digestion LC-MS and MALDI-TOF as rabbit α- and β-chain hemoglobin (undigested) and transtherytin. Lysozyme transcript was detected in midgut cell extracts but the peptide was not. Studies done on other tick species demonstrated that hemoglobin digestion resulted in antimicrobial fragments. Antimicrobial hemoglobin fragments (including fragments larger than any reported previously) also were found in D. variabilis, as well as ubiquitin, a peptide known to occur as part of an antimicrobial complex in vertebrate leukocytes. In addition, we noted that Borrelia burgdorferi spirochetes were not lysed in the midgut lumen, which would be expected if defensin and lysozyme were active in this location. In this respect, the midgut\u27s response to microbial challenge differs from that of the hemolymph. In summary, the midgut\u27s antimicrobial activity appears to be primarily a byproduct of hemoglobin digestion rather than expression of immune peptides and proteins

Glass Capillary Tube Feeding: A Method for Infecting Nymphal Ixodes scapularis (Acari: Ixodidae) with the Lyme Disease Spirochete Borrelia burgdorferi

We evaluated an artificial capillary feeding method to infect nymphal Ixodes scapularis (Say) ticks with Borrelia burgdorferi, the causative agent of Lyme disease. Thirty to 70% of the nymphs were infected after feeding for 2.5 h from glass capillary tubes filled with a solution of spirochetes. Capillary infection was stable and persisted in the nymphs for at least 10 d after feeding. Capillary feeding also maintained natural vector competence patterns because I. scapularis ticks acquired infection unlike Dermacentor variablis (Say), which did not become infected. Capillary infected I. scapularis nymphs were capable of transmitting the infection to naive mice although not as efficiently as naturally infected nymphs. The capillary infection method is convenient and is a better alternative to syringe inoculation as a means of infecting animals with B. burgdorferi

Rickettsia Phylogenomics: Unwinding the Intricacies of Obligate Intracellular Life

BACKGROUND: Completed genome sequences are rapidly increasing for Rickettsia, obligate intracellular alpha-proteobacteria responsible for various human diseases, including epidemic typhus and Rocky Mountain spotted fever. In light of phylogeny, the establishment of orthologous groups (OGs) of open reading frames (ORFs) will distinguish the core rickettsial genes and other group specific genes (class 1 OGs or C1OGs) from those distributed indiscriminately throughout the rickettsial tree (class 2 OG or C2OGs). METHODOLOGY/PRINCIPAL FINDINGS: We present 1823 representative (no gene duplications) and 259 non-representative (at least one gene duplication) rickettsial OGs. While the highly reductive (approximately 1.2 MB) Rickettsia genomes range in predicted ORFs from 872 to 1512, a core of 752 OGs was identified, depicting the essential Rickettsia genes. Unsurprisingly, this core lacks many metabolic genes, reflecting the dependence on host resources for growth and survival. Additionally, we bolster our recent reclassification of Rickettsia by identifying OGs that define the AG (ancestral group), TG (typhus group), TRG (transitional group), and SFG (spotted fever group) rickettsiae. OGs for insect-associated species, tick-associated species and species that harbor plasmids were also predicted. Through superimposition of all OGs over robust phylogeny estimation, we discern between C1OGs and C2OGs, the latter depicting genes either decaying from the conserved C1OGs or acquired laterally. Finally, scrutiny of non-representative OGs revealed high levels of split genes versus gene duplications, with both phenomena confounding gene orthology assignment. Interestingly, non-representative OGs, as well as OGs comprised of several gene families typically involved in microbial pathogenicity and/or the acquisition of virulence factors, fall predominantly within C2OG distributions. CONCLUSION/SIGNIFICANCE: Collectively, we determined the relative conservation and distribution of 14354 predicted ORFs from 10 rickettsial genomes across robust phylogeny estimation. The data, available at PATRIC (PathoSystems Resource Integration Center), provide novel information for unwinding the intricacies associated with Rickettsia pathogenesis, expanding the range of potential diagnostic, vaccine and therapeutic targets

New Tick Defensin Isoform and Antimicrobial Gene Expression in Response to Rickettsia montanensis Challenge

Recent studies aimed at elucidating the rickettsia-tick interaction have discovered that the spotted fever group rickettsia Rickettsia montanensis, a relative of R. rickettsii, the etiologic agent of Rocky Mountain spotted fever, induces differential gene expression patterns in the ovaries of the hard tick Dermacentor variabilis. Here we describe a new defensin isoform, defensin-2, and the expression patterns of genes for three antimicrobials, defensin-1 (vsnA1), defensin-2, and lysozyme, in the midguts and fat bodies of D. variabilis ticks that were challenged with R. montanensis. Bioinformatic and phylogenetic analyses of the primary structure of defensin-2 support its role as an antimicrobial. The tissue distributions of the three antimicrobials, especially the two D. variabilis defensin isoforms, are markedly different, illustrating the immunocompetence of the many tissues that R. montanensis presumably invades once acquired by the tick. Antimicrobial gene expression patterns in R. montanensis-challenged ticks suggest that antimicrobial genes play a role during the acquisition-invasion stages in the tick