Microbial Invasion vs. Tick Immune Regulation

Ticks transmit a greater variety of pathogenic agents that cause disease in humans and animals than any other haematophagous arthropod, including Lyme disease, Rocky Mountain spotted fever, human granulocytic anaplasmosis, babesiosis, tick-borne encephalitis, Crimean Congo haemorhagic fever, and many others (Gulia-Nuss et al., 2016). Although diverse explanations have been proposed to explain their remarkable vectorial capacity, among the most important are their blood feeding habit, their long term off-host survival, the diverse array of bioactive molecules that disrupt the host\u27s natural hemostatic mechanisms, facilitate blood flow, pain inhibitors, and minimize inflammation to prevent immune rejection (Hajdušek et al., 2013). Moreover, the tick\u27s unique intracellular digestive processes allow the midgut to provide a relatively permissive microenvironment for survival of invading microbes. Although tick-host-pathogen interactions have evolved over more than 300 million years (Barker and Murrell, 2008), few microbes have been able to overcome the tick\u27s innate immune system, comprising both humoral and cellular processes that reject them. Similar to most eukaryotes, the signaling pathways that regulate the innate immune response, i.e., the Toll, IMD (Immunodeficiency) and JAK-STAT (Janus Kinase/Signal Transducers and Activators of Transcription) also occur in ticks (Gulia-Nuss et al., 2016). Recognition of pathogen-associated molecular patterns (PAMPs) on the microbial surface triggers one or the other of these pathways. Consequently, ticks are able to mount an impressive array of humoral and cellular responses to microbial challenge, including anti-microbial peptides (AMPs), e.g., defensins, lysozymes, microplusins, etc., that directly kill, entrap or inhibit the invaders. Equally important are cellular processes, primarily phagocytosis, that capture, ingest, or encapsulate invading microbes, regulated by a primordial system of thioester-containing proteins, fibrinogen-related lectins and convertase factors (Hajdusek et al., 2013). Ticks also express reactive oxygen species (ROS) as well as glutathione-S-transferase, superoxide dismutase, heat shock proteins and even protease inhibitors that kill or inhibit microbes. Nevertheless, many tick-borne microorganisms are able to evade the tick\u27s innate immune system and survive within the tick\u27s body. The examples that follow describe some of the many different strategies that have evolved to enable ticks to transmit the agents of human and/or animal disease

Development of microsatellites for genetic analyses and population assignment of the cat flea (Siphonaptera: Pulicidae)

Cat fleas, Ctenocephalides felis (Bouch) (Siphonaptera: Pulicidae), are common ectoparasites of companion animals that negatively impact their hosts directly by causing dermatitis and blood loss during feeding and indirectly through the potential transmission of disease causing agents. We isolated and characterized seven novel microsatellite loci from a partial genomic library of the cat flea enriched for di-, tri-, and tetranucleotide repeats. We screened these loci in cat fleas from two laboratory colonies and one wild-caught population collected at a temporary animal shelter (Parker coliseum) in Baton Rouge, LA. Six loci were polymorphic, with two to 15 alleles per locus and an average observed heterozygosity of 0.21 across populations. Although the two laboratory cat flea colonies were isolated from each other for many years, they did not significantly differ in their genotypic composition. The cat flea population from Parker coliseum was genetically different from the laboratory colonies, but also showed high degrees of inbreeding. Multilocus genotypes of the polymorphic loci were sufficient to assign over 85% of cat fleas to their population of origin. Genetic markers for flea population identity will allow further studies to examine the origins and movement of cat fleas with important genetic traits such as insecticide resistance or pathogen susceptibility. The use of microsatellites also could determine if there are host-specific strains of cat fleas and add insight into the development of the different subspecies of C. felis. © 2010 Entomological Society of America

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

Molecular Characterization and Tissue-Specific Gene Expression of Dermacentor variabilis α-catenin in Response to Rickettsial Infection

Alpha catenin is a cytoskeleton protein that acts as a regulator of actin rearrangement by forming an E-cadherin adhesion complex. In Dermacentor variabilis, a putative α-catenin (Dvα-catenin) was previously identified as differentially regulated in ovaries of ticks chronically infected with Rickettsia montanensis. To begin characterizing the role(s) of Dvα-catenin during rickettsial infection, the full-length Dvα-catenin cDNA was cloned and analysed. Comparative sequence analysis demonstrates a 3069-bp cDNA with a 2718-bp open reading frame with a sequence similar to Ixodes scapularis α-catenin. A portion of Dvα-catenin is homologous to the vinculin-conserved domain containing a putative actin-binding region and β-catenin-binding and -dimerization regions. Quantitative reverse-transcription PCR analysis demonstrated that Dvα-catenin is predominantly expressed in tick ovaries and is responsive to tick feeding. The tissue-specific gene expression analysis of ticks exposed to Rickettsia demonstrates that Dvα-catenin expression was significantly downregulated 12 h after exposure to R. montanensis, but not in Rickettsia amblyommii-exposed ovaries, compared with Rickettsia-unexposed ticks. Studying tick-derived molecules associated with rickettsial infection will provide a better understanding of the transmission dynamics of tick-borne rickettsial diseases

Comparative microbiota of Rickettsia felis-uninfected and -infected colonized cat fleas, Ctenocephalides felis

Fleas serve as arthropod vectors for several emerging and re-emerging infectious disease causing agents including, Rickettsia felis. Although the prevalence of R. felis infection in colonies of fleas has been examined, the influence of the R. felis infection on flea microbiota has not been investigated. We identified three colonies of cat fleas, Ctenocephalides felis, with varying prevalence of R. felis infection (Louisiana State University (LSU), 93.8; Professional Laboratory and Research Services Inc. (PLRS), 16.4; Elward II (EL), 0) and subsequently utilized polymerase chain reaction amplification, restriction fragment length polymorphism analysis and sequencing of the 1.4-kb portions of 16S rRNA genes to examine the diversity of bacteria in the flea populations. A total of 17 different bacterial 16S rRNA gene sequences were identified among the C. felis colonies. The prevalence of two Wolbachia species that were identified in each flea colony differed between colonies and R. felis-uninfected and -infected fleas. Species richness was unchanged among the R. felis-uninfected (LSU, PLRS and EL colonies) and -infected (LSU and PLRS colonies) fleas; however, between R. felis-uninfected and -infected fleas within both the LSU and PLRS colonies, R. felis-uninfected fleas have greater species richness. Diversity indices did not identify a difference in diversity between any of the flea samples. The interaction of endosymbionts within arthropods can widely impact the dissemination of vertically transmitted pathogenic bacteria; and the reciprocal may be true. These results suggest that carriage of R. felis has an impact on the richness of flea microbiota. © 2007 International Society for Microbial Ecology All rights reserved

A Tangled Web: Origins of Reproductive Parasitism

While typically a flea parasite and opportunistic human pathogen, the presence of Rickettsia felis (strain LSU-Lb) in the non-blood- feeding, parthenogenetically reproducing booklouse, Liposcelis bostrychophila, provides a system to ascertain factors governing not only host transitions but also obligate reproductive parasitism (RP). Analysis of plasmid pLbAR, unique to R. felis str. LSU-Lb, revealed a toxin–antitoxin module with similar features to prophage-encoded toxin–antitoxin modules utilized by parasitic Wolbachia strains to induce another form of RP, cytoplasmic incompatibility, in their arthropod hosts. Curiously, multiple deubiquitinase and nuclease domains of the large (3,841 aa) pLbAR toxin, as well the entire antitoxin, facilitated the detection of an assortment of related proteins from diverse intracellular bacteria, including other reproductive parasites. Our description of these remarkable components of the intracellular mobilome, including their presence in certain arthropod genomes, lends insight on the evolution of RP, while invigo- rating research on parasite-mediated biocontrol of arthropod-borne viral and bacterial pathogens

Prevalence and Infection Load Dynamics of Rickettsia felis in Actively Feeding Cat Fleas

Background: Rickettsia felis is a flea-associated rickettsial pathogen recurrently identified in both colonized and wild-caught cat fleas, Ctenocephalides felis. We hypothesized that within colonized fleas, the intimate relationship between R. felis and C. felis allows for the coordination of rickettsial replication and metabolically active periods during flea bloodmeal acquisition and oogenesis. Methodology/Principal Findings: A quantitative real-time PCR assay was developed to quantify R. felis in actively feeding R. felis-infected fleas. In three separate trials, fleas were allowed to feed on cats, and a mean of 3.9610 6 R. felis 17-kDa gene copies was detected for each flea. A distinct R. felis infection pattern was not observed in fleas during nine consecutive days of bloodfeeding. However, an inverse correlation between the prevalence of R. felis-infection, which ranged from 96 % in Trial 1 to 35 % in Trial 3, and the R. felis-infection load in individual fleas was identified. Expression of R. felis-infection load as a ratio of R. felis/C. felis genes confirmed that fleas in Trial 3 had significantly greater rickettsial loads than those in Trial 1. Conclusion/Significance: Examining rickettsial infection dynamics in the flea vector will further elucidate the intimate relationship between R. felis and C. felis, and facilitate a more accurate understanding of the ecology and epidemiology of R. felis transmission in nature

Geographic Association of Rickettsia felis-Infected Opossums with Human Murine Typhus, Texas

Application of molecular diagnostic technology in the past 10 years has resulted in the discovery of several new species of pathogenic rickettsiae, including Rickettsia felis. As more sequence information for rickettsial genes has become available, the data have been used to reclassify rickettsial species and to develop new diagnostic tools for analysis of mixed rickettsial pathogens. R. felis has been associated with opossums and their fleas in Texas and California. Because R. felis can cause human illness, we investigated the distribution dynamics in the murine typhus–endemic areas of these two states. The geographic distribution of R. felis-infected opossum populations in two well-established endemic foci overlaps with that of the reported human cases of murine typhus. Descriptive epidemiologic analysis of 1998 human cases in Corpus Christi, Texas, identified disease patterns consistent with studies done in the 1980s. A close geographic association of seropositive opossums (22% R. felis; 8% R. typhi) with human murine typhus cases was also observed

Interaction of Rickettsia felis with histone H2B facilitates the infection of a tick cell line

Haematophagous arthropods are the primary vectors in the transmission of Rickettsia, yet the molecular mechanisms mediating the rickettsial infection of arthropods remain elusive. This study utilized a biotinylated protein pull-down assay together with LC-MS/MS to identify interaction between Ixodes scapularis histone H2B and Rickettsia felis. Co-immunoprecipitation of histone with rickettsial cell lysate demonstrated the association of H2B with R. felis proteins, including outer-membrane protein B (OmpB), a major rickettsial adhesin molecule. The rickettsial infection of tick ISE6 cells was reduced by approximately 25 % via RNA-mediated H2B-depletion or enzymic treatment of histones. The interaction of H2B with the rickettsial adhesin OmpB suggests a role for H2B in mediating R. felis internalization into ISE6 cells