Here, we demonstrated the utilization of next generation sequencing (NGS) for high-throughput sequencing to identify R. canadensis in Ixodes persulcatus. In previous surveillances during 2011–2021 in China, high percentage of tick samples were PCR-positive for Rickettsia DNA (reviewed by Tian et al. 2022, Shao et al., 2021). Instead of using Sanger sequencing to characterize Rickettsia with specific target genes which would require large amounts of time to assemble and analyze each individual sequence, we applied the NGS transcriptomic technology to sequence R. canadensis for functional genes including rpoB-rpoC-rpoC/160KDa-leucyl aminopeptidase-hypothetical protein-leucyl aminopeptidase-aspartate tRNA ligase, RlmE-Omp 1-Peptidase M50-nusB, dnaK-dnaJ-ChaB family protein-BamD-RecN-Carboxypeptidase M32 simultaneously using Miseq platform in one experimental run. The results of meta transcriptome approaches, consistent to that of 16S rRNA, help much to elucidate the complexities of transmission and circulation of bacteria in the environment by deciphering the active functional genes interacting with host ticks. Our study illustrates another application of NGS for simultaneous characterization of Rickettsia to enable rapid analysis of a large number of samples collected from a surveillance study.
Historically, R. canadensis was originally isolated from Haemaphysalis leporispalustris collected from indicator rabbits near Ottawa in Ontario, Canada, and then be nominated as R. canada in 1967 (McKeil et al. 1967). The proposed name was accepted by the approved list in 1980 (Skerman et al. 1980) and soon were corrected as R. canadensis according to the Rule 12c of the International Code of Nomenclature of Bacteria (ICTB) (Trüper and De'clari, 1997). Since discovered, R. canadensis has also been isolated from ixodid ticks across countries even continents, including Canada, the United States, Russia, Japan, China, South Korea and as well. H. japonica (Russia, Japan) (Golovljova and Tikunova, 2018), H. flava (Japan), H. leporispalustris and Dermacentor andersoni (USA) (Philip et al. 1982)d longicornis (China) (Qin et al. 2019) were also found naturally infected with R. canadensis, which suggested its wide geographic distribution and broad arthropod host spectrum. Our results also demonstrated that R. canadensis prevailed in I. persulcatus in Zhaluntun city, Inner Mongolia Autonomous Region, and Mudanjiang city, Heilongjiang province, China, with strong evidences of 3 arrays of 16 functional genes. Similarly, Tian and his colleagues (Tian et al. 2022) also reported that I. persulcatus from Yakeshi, Inner Mongolia Autonomous Region and Suifenhe, Heilongjiang province carrying R. canadensis DNA amplicons for atpD (ABV73937.1), coxB (ABV73605.1), ftsZ (ABV73606.1), gltA (ABV73997.1), groEL (ABV 732 69.1) and sucA (ABV73134.1) genes. The two independent discoveries strongly indicated that R. canadensis definitely occurs in I. persulcatus in China. Moreover, many efforts to unravel the pathogenicity of R. canadensis were performed including rickettsia isolations (McKeil et al. 1967), ultramicroscopic observations(Avakian et al. 1973), animal inoculations (Burgdorfer and Brinton 1970), experimental transmissions via ticks (Burgdorfer 1968, Balayeva et al. 1977) and epidemiological surveys in human populations. As results, serologic evidences were achieved by the presences of R. canadensis antibody in a febrile male patient (Bozeman et al. 1970), victims suffer from Rocky Mountain Spotted Fever-like disease (Ignatovich et al. 1977) and acute cerebral vasculitis (Ignatovich et al. 1976). In an epidemiology survey for tick borne pathogens among forest rangers in Inner Monogolia Autonomous Region of China, outer membrane protein (ompB) gene of R. canadensis had also been sequenced from a male asymptotic forest ranger (MH549232.1 directly submitted). However, till to now, the pathogenicity of R. canadensis is still controversy due to the possible cross-reaction against antibodies from other pathogenic rickettsia species and shortage of direct association of clinical manifestations and the infection of R. canadensis in human beings.
As shown in several virulent strains of pathogenic rickettsiae, the line between pathogenic bacteria and endosymbionts is not well defined. The pathogenic and endosymbiotic nature of rickettsiae may have evolved through different scenarios. First, a loss of pathogenicity, for example, by R. peacockii, a strictly endosymbiotic Rickettsia that is closely related to the severely pathogenic R. rickettsii. Various deletions and mutations were observed in the genome of R. peacockii by transposon recombination that eliminated its pathogenic ability (Felsheim et al. 2009; Gillespie et al. 2012). The representative gene, rickA, responsible for actin tail polymerization in cell-to-cell mobility is found loss of function in R. peacockii due to the insertion of a mobile element (Simser et al. 2005), which partially explain why R. peacockii is not horizontally transmitted and non-pathogenic to vertebrates. Meanwhile, outer membrane proteins (ompA or ompB), a crucial virulent factor for autophagy evasion of Rickettsia, also offer useful clues helping to infer pathogenicity with reduced numbers of functional membrane components (antigenic epitopes) and rapid evolution under the positive selection to confront against host immune response (Jiggins et al. 2002). R. canadensis and R. typhi may also define a similar situation under the same hypothesis. Second, gain of pathogenicity via horizontal gene transfer (HGT) mechanisms among different endosymbionts. The repeated occurrences of HGT provide much opportunities to evolve into novel bacterial phenotypes, as in Coxiella burnetii, which infects vertebrate cells, was assumed to originate from non-pathogenic Coxiella progenitor by gain pathogenic genes from other endosymbionts (Duron et al. 2017). Therefore, researches on genes identical to the pathogenicity of R. canadensis and its partners would ultimately serve as clues as to whether other Rickettsia can potentially cause disease in vertebrates and elucidate the specific adaptations that Rickettsia use to exploit vertebrate hosts and non-specific responses that may cause disease. Generally, genus Rickettsia clearly fits with a biphasic model of genomic evolution, including genomic reduction by pseudogenization and loss of genes and/or plasmids as well as genomic expansion by gene amplification and/or plasmid multiplication (Karkouri et al. 2022). The genome expansion identified in the SFG and BG species originated more from gene duplications and proliferations of both orthologous and/or xenologous genes. In contract, the genome degradations or reductions were confirmed in TG and CG rickettsiae with smaller genomes, which is known to have occurred during the transition of bacteria from a free-living to obligate intracellular lifestyle under more relaxed selective pressure. And thus, R. canadensis exploits great accessibility to host cell resources and other environmental factors as possible for survival. However, although slight increase in virulence has been observed associated with the reductive evolutionary process in CG rickettsiae, adaptive substitutions of core genes of R. canadensis contributed to the minor changes in pathogenicity and morbid symptoms requires further investigation. Furthermore, it should also be noted that Rickettsia-infected Dermacentor ticks showed more motility than uninfected ones (Kagemann and Clay 2013). Higher motility associated to host-seeking behavior in ticks indirectly influences infection risk by increasing the rates of tick bites and pathogen transmission. Whether the infection of R. canadensis in I. persulcatus improve the motility of host ticks over ecological timescales and the potential roles epidemiologically relevant in the spread of R. canadensis and other tick borne pathogens also remain to be determined.