High Diversity of Tick-associated Microbiota from Five Tick Species in Yunnan, China

Ticks are obligate blood-sucking vectors for multiple zoonotic diseases. In this study, tick samples were collected from Yunnan Province, China, which is well-known as the “Global Biodiversity Hotspot” in the world. This study aimed to clarify the microbial populations, including pathogens, associated with ticks and to identify the diversity of tick-borne microbiota in this region. The 16S rRNA full-length sequencing from pooled tick DNA samples and PCR amplification of pathogenic genera from individual samples were performed to understand tick-associated microbiota in this region. A total of 191 adult ticks of 5 tick species were included and revealed 11 phyla and 126 genera bacteria, including pathogenic Anaplasma , Ehrlichia , Candidatus Neoehrlichia, Rickettsia , Borrelia , and Babesia . Further identification suggested that Rickettsia sp. YN01 was a variant strain of Rickettsia spp. IG-1, but Rickettsia sp. YN02 and Rickettsia sp. YN03, were potentially two new SFGR species. This study revealed the complexity of ecological interactions between host and microbe and provided insight for the biological control of ticks. A high microbial diversity in ticks from Yunnan was identified, and more investigation should be undertaken to elucidate the pathogenicity in the area

Wide Distribution and Genetic Diversity of Babesia microti in Small Mammals from Yunnan Province, Southwestern China.

BACKGROUND:Babesia, usually found in wild and domestic mammals worldwide, have recently been responsible for emerging malaria-like zoonosis in infected patients. Human B. microti infection has been identified in China, primarily in the Southwest along the Myanmar border but little direct surveillance of B. microti infection in rodents has been carried out here (Yunnan province). In this region, a diverse topographic range combined with tropical moisture sustains a high biodiversity of small mammals, which might play important role on Babesia transmission. METHODS:Small mammals were captured in 141 sample locations from 18 counties located Yunnan Province, and screened for B. microti-like parasites infection by a nested PCR to target 18S rRNA gene of Babesia, plus directly sequencing for positive samples. Univariate and multivariate forward stepwise logistic regression analysis was used to access the association between infections and some related risk factors. RESULTS:Infection with Babesia microti was confirmed in 2.4% (53/ 2204) of small mammals. Significant differences in prevalence rates of B. microti were observed based on variations in forest, agricultural, and residential landscapes. Furthermore, adult small mammals had higher prevalence rates than younger, pubertal mammals. The near full-length 18S rRNA gene revealed that there were two types of B. microti, Kobe and Otsu, which demonstrate the genetic diversity and regional distribution. CONCLUSIONS:There exists a wide distribution and genetic diversity of endemic B. microti in Southwestern China, warranting further investigations and monitoring of clinical disease in individuals presenting with Babesia like symptoms in these areas

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

Neighbor-joining phylogenetic tree based on a comparison of <i>Babesia microti</i> 18S rRNA gene sequences obtained from Yunnan small mammals with <i>Babesia microti</i> reference strains.

<p><i>Babesia felis</i> was included as the outgroup. The number on each branch shows the percent occurrence in 1,000 bootstrap replicates. Black circles stood for novel sequences identified in this study.</p

Risk factors related to <i>B</i>. <i>microti</i> based on univariate analyses.

<p>Risk factors related to <i>B</i>. <i>microti</i> based on univariate analyses.</p

Risk factors related to <i>B</i>. <i>microti</i> based on multivariate logistic regression.

<p>Risk factors related to <i>B</i>. <i>microti</i> based on multivariate logistic regression.</p

Wide Distribution and Genetic Diversity of <i>Babesia microti</i> in Small Mammals from Yunnan Province, Southwestern China

<div><p>Background</p><p><i>Babesia</i>, usually found in wild and domestic mammals worldwide, have recently been responsible for emerging malaria-like zoonosis in infected patients. Human <i>B</i>. <i>microti</i> infection has been identified in China, primarily in the Southwest along the Myanmar border but little direct surveillance of <i>B</i>. <i>microti</i> infection in rodents has been carried out here (Yunnan province). In this region, a diverse topographic range combined with tropical moisture sustains a high biodiversity of small mammals, which might play important role on <i>Babesia</i> transmission.</p><p>Methods</p><p>Small mammals were captured in 141 sample locations from 18 counties located Yunnan Province, and screened for <i>B</i>. <i>microti</i>-like parasites infection by a nested PCR to target 18S rRNA gene of <i>Babesia</i>, plus directly sequencing for positive samples. Univariate and multivariate forward stepwise logistic regression analysis was used to access the association between infections and some related risk factors.</p><p>Results</p><p>Infection with <i>Babesia microti</i> was confirmed in 2.4% (53/ 2204) of small mammals. Significant differences in prevalence rates of <i>B</i>. <i>microti</i> were observed based on variations in forest, agricultural, and residential landscapes. Furthermore, adult small mammals had higher prevalence rates than younger, pubertal mammals. The near full-length 18S rRNA gene revealed that there were two types of <i>B</i>. <i>microti</i>, Kobe and Otsu, which demonstrate the genetic diversity and regional distribution.</p><p>Conclusions</p><p>There exists a wide distribution and genetic diversity of endemic <i>B</i>. <i>microti</i> in Southwestern China, warranting further investigations and monitoring of clinical disease in individuals presenting with <i>Babesia</i> like symptoms in these areas.</p></div

Prevalence of <i>B</i>. <i>microti</i> in small mammals of different species.

<p>Prevalence of <i>B</i>. <i>microti</i> in small mammals of different species.</p

Prevalence of <i>B</i>. <i>microti</i> in small mammals from different survey sites.

<p>Prevalence of <i>B</i>. <i>microti</i> in small mammals from different survey sites.</p