Genetic diversity and prevalence of emerging Rickettsiales in Yunnan Province: a large-scale study.

BACKGROUND: Rickettsia and related diseases have been identified as significant global public health threats. This study involved comprehensive field and systematic investigations of various rickettsial organisms in Yunnan Province. METHODS: Between May 18, 2011 and November 23, 2020, field investigations were conducted across 42 counties in Yunnan Province, China, encompassing small mammals, livestock, and ticks. Preliminary screenings for Rickettsiales involved amplifying the 16S rRNA genes, along with additional genus- or species-specific genes, which were subsequently confirmed through sequencing results. Sequence comparisons were carried out using the Basic Local Alignment Search Tool (BLAST). Phylogenetic relationships were analyzed using the default parameters in the Molecular Evolutionary Genetics Analysis (MEGA) program. The chi-squared test was used to assess the diversities and component ratios of rickettsial agents across various parameters. RESULTS: A total of 7964 samples were collected from small mammals, livestock, and ticks through Yunnan Province and submitted for screening for rickettsial organisms. Sixteen rickettsial species from the genera Rickettsia, Anaplasma, Ehrlichia, Neoehrlichia, and Wolbachia were detected, with an overall prevalence of 14.72%. Among these, 11 species were identified as pathogens or potential pathogens to humans and livestock. Specifically, 10 rickettsial organisms were widely found in 42.11% (24 out of 57) of small mammal species. High prevalence was observed in Dremomys samples at 5.60%, in samples from regions with latitudes above 4000 m or alpine meadows, and in those obtained from Yuanmou County. Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis were broadly infecting multiple genera of animal hosts. In contrast, the small mammal genera Neodon, Dremomys, Ochotona, Anourosorex, and Mus were carrying individually specific rickettsial agents, indicating host tropism. There were 13 rickettsial species detected in 57.14% (8 out of 14) of tick species, with the highest prevalence (37.07%) observed in the genus Rhipicephalus. Eight rickettsial species were identified in 2375 livestock samples. Notably, six new Rickettsiales variants/strains were discovered, and Candidatus Rickettsia longicornii was unambiguously identified. CONCLUSIONS: This large-scale survey provided further insight into the high genetic diversity and overall prevalence of emerging Rickettsiales within endemic hotspots in Yunnan Province. The potential threats posed by these emerging tick-borne Rickettsiales to public health warrant attention, underscoring the need for effective strategies to guide the prevention and control of emerging zoonotic diseases in China.

Systematic investigation of the Borrelia miyamotoi spirochetes in ticks, wildlife and domestic animal hosts in Yunnan province, Southwest China.

Background: Borrelia miyamotoi is a spirochete species transmitted via hard ticks. Following its discovery in Japan, this pathogen has been detected around the world, and is increasingly confirmed as a human pathogen causing febrile disease, namely relapsing fever. Its presence has been confirmed in the Northeast China. However, there is little information regarding the presence of B. miyamotoi and other hard-tick-borne relapsing fever spirochetes in southern China including Yunnan province, where tick and animal species are abundant and many people both inhabit and visit for recreation. Methods: For the present study, we collected samples of ticks, wildlife, and domestic animal hosts from different counties in Yunnan province. Nucleic acids from samples were extracted, and the presence of B. miyamotoi and other relapsing fever spirochetes was confirmed using polymerase chain reaction (PCR) for the 16S rRNA specific target gene fragment. The positive samples were then amplified for partial genome of the flaB and glpQ genes. Statistical differences in its distribution were analyzed by SPSS 20 software. Sequence of partial 16S rRNA, flaB and glpQ genome were analyzed and phylogenetic trees were constructed. Results: A total of 8260 samples including 2304 ticks, 4120 small mammals and 1836 blood of domestic animal hosts were collected for screening for infection of B. miyamotoi and other relapsing fever spirochetes. Cattle and sheep act as the main hosts and Rhipicephalus microplus, Haemaphysalis nepalensis, H. kolonini and Ixodes ovatus were identified as the important vector host with high prevalence or wide distribution. Only one Mus caroli (mouse) and one Sorex alpinus (shrew) were confirmed positive for relapsing fever spirochetes. Evidence of vertical transmission in ticks was also confirmed. Two known strains of B. miyamotoi and one novel relapsing fever spirochetes, B. theileri-like agent, were confirmed and described with their host adaptation, mutation, and potential risk of spreading and spillover for human beings. Conclusions: Our results provide new evidence of relapsing fever spirochetes in vector and animal hosts in Yunnan province based on large sample sizes, and offer guidance on further investigation, surveillance and monitoring of this pathogen.

Coinfection of Two Rickettsia Species in a Single Tick Species Provides New Insight into Rickettsia-Rickettsia and Rickettsia-Vector Interactions.

Rickettsiae are obligate intracellular bacteria that can cause life-threatening illnesses. There is an ongoing debate as to whether established infections by one Rickettsia species preclude the maintenance of the second species in ticks. Here, we identified two Rickettsia species in inoculum from Haemaphysalis montgomeryi ticks and subsequently obtained pure isolates of each species by plaque selection. The two isolates were classified as a transitional group and spotted fever group rickettsiae and named Rickettsia hoogstraalii str CS and Rickettsia rhipicephalii str EH, respectively. The coinfection of these two Rickettsia species was detected in 25.6% of individual field-collected H. montgomeryi. In cell culture infection models, R. hoogstraalii str CS overwhelmed R. rhipicephalii str EH with more obvious cytopathic effects, faster plaque formation, and increased cellular growth when cocultured, and R. hoogstraalii str CS seemed to polymerize actin tails differently from R. rhipicephalii str EH in vitro. This work provides a model to investigate the mechanisms of both Rickettsia-Rickettsia and Rickettsia-vector interactions. IMPORTANCE The rickettsiae are a group of obligate intracellular Gram-negative bacteria that include human pathogens causing an array of clinical symptoms and even death. There is an important question in the field, that is whether one infection can block the superinfection of other rickettsiae. This work demonstrated the coinfection of two Rickettsia species in individual ticks and further highlighted that testing the rickettsial competitive exclusion hypothesis will undoubtedly be a promising area as methods for bioengineering and pathogen biocontrol become amenable for rickettsiae.