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. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40249-024-01213-4.

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.Over the past three decades, advances in molecular diagnostic techniques have led to the rapid and precise identification of emerging pathogens among tick-associated rickettsial agents, as well as some Candidatus species of rickettsial agents worldwide [14][15][16][17][18][19].In particular, novel rickettsioses and newly recognized natural foci often emerge unexpectedly.The rising popularity of ecotourism, extensive global travel, and the frequent migration of wild animal populations along with parasitic ticks have significantly contributed to the increasing incidence of tick-associated rickettsioses.Therefore, addressing this issue and reducing the burden of human rickettsioses presents significant challenges for public health authorities, physicians, veterinarians, and scientists.
In China, numerous reports have documented the distribution and co-circulation of Rickettsiales bacteria among ticks, wild animals, livestock, and febrile patients [20][21][22].Yunnan Province, located in Southwest China and bordering the Indo-Chinese peninsula, is characterized by diverse eco-climatic zones and is recognized as a biodiversity hotspot.This region is of unique medical significance for assessing potential spillover vector-borne zoonoses and the emergence of new pathogens.Previous surveys have highlighted the high diversity of Rickettsiales in limited tick and small mammals samples collected from Yunnan [20,[23][24][25][26].It is speculated that the host and vector species of tick-associated Rickettsiales in Yunnan Province are abundant and widely dispersed.In this study, extensive field and systematic investigations of multiple rickettsial species were conducted using molecular detection methods to reveal the true nature of Rickettsiales infections and their potential threats to public health.

Study sites and sample collection
Between May 18, 2011 and November 23, 2020, field investigations covering small wildlife mammals, domestic animal hosts, and tick vectors were conducted across 42 counties of Yunnan Province, encompassing various geographical terrains such as alpine meadows, cultivated lands, bushes, woodlands, coniferous forests, broadleaved forests, mixed forests, bamboo forests, residential areas, and dry-hot valleys, spanning an altitude gradient ranging from 383 to 4000 m above sea level.
Small mammals were sampled using the baited snap night-trap method.Morphological identification of captured animals was performed by experienced biologists and subsequently confirmed through sequence analysis of partial cytochrome oxidase I (COI) gene of suspected rodent species, followed by aseptic dissection to obtain liver and spleen tissue samples [27].Livestock were sampled for whole blood collection via jugular vein puncture using EDTA anticoagulant tubes.Domestic mammal-associated ticks were collected by manual scratching, while host-seeking ticks were obtained through flag-sweeping on vegetation at the same sampling sites.Morphological identification of ticks was conducted by trained entomologists and subsequently validated using molecular evidence from COI genes [28].All collected samples, including blood, tissues, and ticks, were transported to the laboratory under cold-chain conditions and stored at -80 °C until further processed.

DNA extraction, PCR and sequencing
DNA extraction from host blood samples and small mammal tissue samples was conducted based on the instructions provided by the manufacturer with the TIANamp Genomic DNA Kit (TIANGEN, Cat.No. 4992199).The identified ticks underwent surface sterilization with 70% alcohol, followed by drying and resin coating in PBS before being crushed.Buffer ATL (a tissue lysis buffer) and protease K were added to the homogenized liquid to facilitate tissue lysis.Subsequently, after centrifugation in a water bath, buffer ATL and anhydrous ethanol were added for DNA extraction.The DNA from individual ticks was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Cat.No. 69504), and the extracted DNA was stored in a freezer set at -80 °C for future use.

Sequence comparisons and phylogenetic analyses
Sequence comparisons were conducted using the Basic Local Alignment Search Tool (BLAST) available on the NCBI website (https:// blast.ncbi.nlm.nih.gov/ Blast.cgi) to analyze species and gene types.All sequences were aligned using Clustal W with default parameters in the Molecular Evolutionary Genetics Analysis (MEGA) program (version 6.0,The Pennsylvania State University, PA, USA).Phylogenetic relationships were inferred using the neighbor-joining method based on 1000 bootstrapped datasets.The statistical support for individual nodes was indicated by posterior probability values.

Statistical analyses
The chi-squared test or Fisher's exact method was used to analyze the diversities and component ratios of rickettsial agents across various parameters, their distribution among different host species, habitats, and other potential influencing factors.Variables with a P value of < 0.05 were deemed statistically significant.All analyses were performed using SPSS (version 17.0, SPSS Inc., Chicago, IL).

Ten species under five genera emerging Rickettsiales in small mammals
In total, 2.59% (112/4330) of sampled individuals and 24 out of 57 (42.11%) species of small mammals were infected with rickettsial agents, primarily concentrated in western counties of Yunnan Province.The highest prevalence of rickettsial agents was observed in Yuanmou County (11.06%), followed by Weixi County (10.00%), with significant differences (P < 0.001) (Table 1).Among the 21 identified genera, the genus Rattus exhibited the highest frequencies of rickettsial agent infections (n = 51), with R. tanezumi having a prevalence of 3.06%.The genus Dremomys had the highest prevalence (5.60%, 1/18).Significant differences in prevalence were observed among genera of small mammals (P = 0.025) (Fig. 2A).Furthermore, the highest  prevalence of rickettsial agents was observed in areas with altitudes above 4000 m (P = 0.004) (Fig. 2B).Additionally, the prevalence of rickettsiae was higher in males than in females (P = 0.004).Small animals collected from alpine meadows exhibited the highest prevalence, followed by those in mixed forests (P = 0.046) (Fig. 2C).
To better understand the potential transmission of rickettsial agents in small mammals, we constructed host-rickettsial correlation networks.Among the small mammals studied, 21 genera were found in 10 families.
Of these, 14 genera in 6 families of small animals were found to be harboring rickettsial agents.Some rickettsial agents were specifically found to infect only one genus, such as Wolbachia sp.strain YL020 in the genus Apodemus, Anaplasma bovis in the genus Niniventer, and Ehrlichia sp.YN04 in the genus Rattus.Conversely, the genera Neodon, Dremomys, Ochatona, and Anourosorex were found to carry A. phagocytophilum, and the genus Mus was detected to host only Wolbachia sp.strain YM011, indicating host tropism, a preference towards certain rickettsia.However, both A. phagocytophilum and Candidatus Neoehrlichia mikurensis (CNM) were detected in more than 7 genera of small animals (Fig. 3), indicating a broad host spectrum.
The constructed phylogenetic tree revealed that the rickettsial species detected in the small mammals clustered into five major groups: Neoehrlichia, Ehrlichia, Anaplasma, Wolbachia, and Rickettsia (Fig. 4).Within the Wolbachia cluster, the sequences identified were significantly different from known species and were categorized into two clades, termed Wolbachia sp.strain YL020 and Wolbachia sp.strain YM011.

High prevalence of eight species emerging Rickettsiales in livestocks
The positivity rate in livestock blood samples collected from 16 counties was 33.68% (800/2375).The highest prevalence was observed in Yingjiang County (81.65%, 89/109), followed by Huaping County (66.02%, 68/103), with significant differences (P < 0.001).Among the four types of livestock sampled, Bovine taurus exhibited the highest number of infected individuals and the highest prevalence of rickettsial agent infections (45.03%, 317/704, P < 0.001).The phylogenetic tree indicated that the rickettsial strains detected in livestock clustered into three distinct groups: Ehrlichia, Anaplasma, and Wolbachia.The predominant species identified were A. ovis, A. marginale, and A. phagocytophilum.Notably, A. phagocytophilum displayed considerable diversity, with at least 3 variants differing by 1 to 30 base pairs, though they were located within the same clade in the phylogenetic tree (Fig. 5).
Both the full-length 16S rRNA gene (Fig. S1A) and the groEL gene of CNM (Fig. S1B) detected in this study were Fig. 3 Host-rickettsial correlation network topology.Rectangles represent the genera of the small mammals; the size of the circle represents the level of rickettsial agents' positivity and the thickness of the straight line represents the level of host infection with rickettsiae Fig. 4 Phylogenetic tree based on partial sequences of 16S rRNA (660 bp) gene of the rickettsiae species derived from small mammals.Phylogenetic analysis was performed using the neighbor-joining method and trees were tested by bootstrapping (1000 replicates) Fig. 5 Phylogenetic tree based on partial sequences of 16S rRNA (660 bp) gene of the rickettsiae species derived from livestock.Phylogenetic analysis was performed using the neighbor-joining method and trees were tested by bootstrapping (1000 replicates) Fig. 6 Phylogenetic tree based on partial sequences of 16S rRNA (660 bp) gene of the rickettsiae species derived from ticks.Phylogenetic analysis was performed using neighbor-joining method and trees were tested by bootstrapping (1000 replicates) remarkably distinct from other sequences detected in China Germany, and Switzerland.Among the three clusters of CNM based on the groEL gene the phylogenetic tree indicated that the present sequences belong to cluster I (southwest) and cluster III (southeast) [37] (Fig. S1B).Although the sequences from Lushui and Weixi were close to the southwest branch (I), they clustered into small branches, indicating they differ from previous ones in Yunnan.
Five Anaplasma-associated pathogens (A.capra, A. ovis, A. bovis, A. marginale, and A. phagocytophilum) were confirmed in samples.The groEL (Fig. S2B) and gltA (Fig. S2C) genes of A. capra revealed 100% homology between Capra aegagrus in Tengchong and patients in Heilongjiang [8].The msp4 gene (Fig. S2D) of A. capra had 99.8% homology, indicating pronounced similarity with the pathogen infecting patients.Variations of A. capra were also detected in Tengchong, Deqin, and Shangri-La (Fig. S2).The phylogenetic tree based on the msp4 and entire 16S rRNA gene (Fig. S3A) of A. ovis formed a cohesive branch closely aligned with reference sequences.Sequences detected in Yunlong consistently clustered within a separate branch, demonstrating a close genetic relationship across different hosts within this geographic region.The gltA gene (Fig. S3C) of A. ovis distributed into two small branches.The groEL gene of A. bovis, A. marginale, and A. phagocytophilum also indicated diversity and distinctive distribution among their host ticks and animals (Fig. S4).The groEL and gltA genes of E. canis clustered in a distinct branch along with other sequences identified in ticks in China, with sister branches from Thailand, the Philippines, Spain, and France.The Dsb and TRP36 genes of E. canis formed unique, singular clades (Fig. S5).

Discussion
We identified the co-circulation of multiple species of Rickettsiales bacteria, including previously uncharacterized species, among small mammals, livestock, and ticks in Yunnan Province.This underscores the enduring importance of Rickettsiales infections in southwestern China and the need for ongoing surveillance of local arthropods, mammals, and humans.
The extensive diversity of tick-associated Rickettsia species globally is well documented, with instances such as R. tillamookensis in the United States and various species found in wild mammals in Morocco and Mauritania [38,39].Advances in phylogenetics and molecular detection techniques have greatly facilitated the identification of numerous novel Rickettsiales species associated with mammals and arthropods [5].In China, new and uncharacterized rickettsial species continue to be discovered such as Rickettsia sp.sw. in Yunnan Province and Ehrlichia sp. in Inner Mongolia [23,40].Recent reports indicate a high diversity of Rickettsia spp.and Ehrlichia spp. in five species of tick from Yunnan Province [26].We further identified six novel strains and one new record in Yunnan along with known Rickettsiales agents, emphasizing the remarkable diversity of Rickettsiales bacteria in ticks, as well as in small mammals and livestock.
There are variations in the reported prevalence of rickettsial agent infections among wild small mammals in different regions such as Southwest China and the Indo-China peninsula (2.02%), Slovakia (19.10%), and Kazakhstan (2.72%) [41][42][43].In our study, the genus Dremomys exhibited a significantly high rate of Rickettsiales positivity among small mammals, while the highest frequency was observed in the genus Rattus, which is widely distributed and commonly found near local residences.Therefore, these two genera should be focused on when monitoring rickettsial infections.Additionally, our observations indicated changes in rickettsial prevalence based on sex and eco-habitats.The higher infection rates in males may be attributed to their more competitive and aggressive behaviors, leading to increased contact and susceptibility to infection.Furthermore, samples collected from alpine meadows revealed higher prevalence rates of Rickettsiales agents, possibly due to the presence of dominant host animals in these habitats.These observations warrant further detailed investigation.In summary, the rickettsial agents detected in these small mammals exhibited distinctive genetic diversity characteristics both interspecifically and intraspecifically.
Moreover, diverse rickettsial species, such as Rickettsia spp., Anaplasma spp., Ehrlichia spp., Neoehrlichia spp., and Wolbachia spp., were identified in 33.68% (800/2375) of livestock and were distributed across almost all surveyed counties.Given the close contact between humans and livestock, this highlights a potentially high risk of human exposure to these rickettsial agents.Among the rickettsial agents detected in livestock, A. phagocytophilum, A. capra, A. platys, and A. bovis variant were confirmed to possess human pathogenicity in earlier studies [7,8,44,45].Additionally, A. bovis and A. bovis-like infections have recently been identified in patients from China and the United States [16,46].Conversely, A. marginale and E. canis are recognized as pathogenic to animals rather than humans [47,48].
Numerous reports in previous literatures have highlighted the risk posed by Rickettsiales, such as A. ovis infections in Iran and Anaplasma spp. in Pakistan [49,50].Our large-scale study confirmed that locations in Yuanmou County, areas at latitudes over 4000 m, and alpine meadow regions exhibited significantly higher Rickettsiales positivity rates among small mammals.Additionally, A. phagocytophilum was frequently detected in small mammals, livestock, and ticks, confirming its widespread distribution in Yunnan Province, particularly in Deqin County, where there is a higher potential for forming transmission chains.
Certain rickettsial species may exhibit specific host or vector associations, indicating host tropism for these agents.While some Rickettsiales are host-specific, others switch hosts or circulate regularly among different hosts, particularly mammals and blood-sucking arthropods [5].Since most emerging human infectious diseases originate from spillover events from vectors or animal hosts, assessing infections among hosts, vectors, and febrile patients in local representative areas is crucial for understanding tick-associated Rickettsiales [4,6,7].Therefore, the characterization and identification of novel Rickettsiales are of significant importance for both human and animal health.
Multigene analysis of E. canis from representative samples revealed a clustering pattern, distinct from international strains, indicating that the E. canis detected in Yunnan Province has unique genetic characteristics.Similarly, the multigene analysis of CNM from Weixi samples revealed close genetic similarities to those from southeastern China, yet notable divergence from strains previously detected in Yunnan, Heilongjiang, and abroad.Based on the previous classification standards for CNM, the CNM from Weixi should be considered distinct variants [44].Our results also indicated that A. ovis was the most prevalent species, with a high positivity rate, widespread distribution, and interspecific differences in the survey region.However, sequences from Yunlong consistently clustered within a separate branch, demonstrating close genetic relationships across different hosts within this specific geographic region.
A comprehensive analysis of multiple genes for our Rickettsia agent, which was closely related to Candidatus R. longicornii, Rickettsia YN03, and R. jingxinensis, revealed that the nomenclature of "Candidatus R. jingxinensis" is not rigorous [51][52][53][54].In fact, "Candidatus R. jingxinensis" and "Candidatus R. longicornii" should be considered synonyms.Based on the rules of species nomenclature, the name "Candidatus R. longicornii" should be accepted as valid, hence it should be referred to as R. longicornii.Similarly, the name Rickettsia YN03 should also be synonymized as R. longicornii, despite the observed degree of variation and considerable diversity.
Our primary limitation was the absence of human case records and samples, which precluded the confirmation of potential infections among local residents.Most study areas are located in remote mountainous regions of Yunnan Province, characterized by a high population of ethnic minorities.These areas are economically underdeveloped, and the local population generally has low awareness of self-protection measures and limited access to medical services.Furthermore, medical services at the grassroots level are inadequate, lacking the capacity for clinical diagnosis and management of emerging tick-associated rickettsioses, leading to frequent missed or misdiagnosed cases.Additionally, the prevalence of tick-associated rickettsioses could negatively impact ecotourism and economic development in Yunnan Province.Therefore, early surveillance of these emerging Rickettsiales among hosts, vectors, and exposed populations in these hotspots is crucial for effective prevention and control.

Conclusions
This large-scale study underscores the genetic diversity and overall prevalence of emerging tick-associated Rickettsiales within biodiversity hotspots in Yunnan Province.These findings reveal the substantial threats posed by emergent Rickettsiales and serve as a crucial reference for the prevention and control of related zoonotic diseases in southwestern China.Given the impact of these emerging Rickettsiales in these hotspots, urgent surveillance and implementation of prevention and control measures for the local human populations is warranted.

Fig. 1
Fig. 1 Distribution of different species of samples and positive samples in different counties in Yunnan Province.Blue, red and green circles represent samples of livestock, small mammals and ticks, " + " represent sites, hosts and vectors with positive results for rickettsiae.The circles size of symbols represents the number of samples

Fig. 2
Fig. 2 Comparison the positivity rate of rickettsiae in different altitudes, hosts, and habitats in Yunnan Province.A Host genus (P = 0.025) B Altitude (P = 0.004) C Habitat types (P = 0.046)

Table 1
Overall prevalence of animals and vectors from different counties with emerging rickettsiae in Yunnan Province -Means no samples were collected