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.

Complete mitogenomes and phylogenetic relationships of Haemaphysalis nepalensis and Haemaphysalis yeni.

The mitochondrial genome may include crucial data for understanding phylogenetic and molecular evolution. We sequenced the complete mitogenome of Haemaphysalis nepalensis and Haemaphysalis yeni for the first time. H. nepalensis and H. yeni's complete mitogenomes were 14,720 and 14,895 bp in size, respectively, and both contained two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and 13 protein-coding genes (PCG). Haemaphysalis nepalensis have one control region (D-loop). The adenine + thymine concentration of the genomes of H. nepalensis and H. yeni was 77.75 and 78.41%, respectively. The codon use pattern and amino acid content of proteins were both observed to be affected by the AT bias. Genes in the mitogenome were organized and located in a comparable manner to previously known genes from Haemaphysalis ticks. Mitochondrial PCGs were used to perform phylogenetic relationships based on the Minimum Evolution (ME) approach using MEGA 7.0 software, the results reveal that H. nepalensis has tight links with H. tibetensis, H. yeni and H. kolonini share a sister group relationship, and that H. nepalensis and H. yeni belong to Haemaphysalis. The results of this study include the following: (i) discovered and supplied new tick records (H. nepalensis) for China, (ii) provided the first complete mitochondrial genome for H. nepalensis and H. yeni and revealed their phylogenetic relationships, and (iii) the features of the mitochondrial genome of H. nepalensis and H. yeni provided more genetic reference for Phylogeography, systematics, and population genetics of the Haemaphysalis species.

High Diversity and Prevalence of Borrelia burgdorferi sensu lato in Wildlife Hosts, Domestic Animals, and Ticks in Yunnan Province, Southwestern China.

Borrelia burgdorferi sensu lato (BBSL), the causative agent of Lyme disease, is commonly found in wild and domestic mammals and ticks worldwide. In China, human cases of Borrelia burgdorferi infections have been identified across a wide geographic range including Yunnan Province, but few studies have examined BBSL in reservoirs and vectors in southwestern China. Here we conducted a thorough and broad-range investigation of BBSL in small mammals, domestic mammals, and ticks collected from 159 sample sites across 42 counties in Yunnan Province. DNA was extracted from spleen tissue of small mammals, blood from domestic mammals, and homogenized ticks. Nested PCR targeting the 5S-23S rRNA intergenic spacer gene of BBSL was used for screening, with amplicons sequenced directly and analyzed using a BLAST algorithm. A total of 8,478 samples were collected, which were composed of 5,044 mammals belonging to 68 species, 1,927 livestock belonging to five species, and 1, 507 ticks belonging to 14 species. BBSL was detected in 147 mammals (2.9%) from 30 different species, 20 of which represent the first reported detection in that species. A total of 52 (2.7%) livestock samples were positive for BBSL, with dogs having the highest detection rate (6.3%, 43/687), and 103 ticks (6.8%) tested positive with high prevalence in Ixodes granulatus (44.2%, 23/52), Haemaphysalis nepalensi (33.3%, 3/9) and Haemaphysalis kolonini (19.0%, 31/163). Sequence analysis revealed six genospecies of BBSL including B. afzelii, B. burgdorferi sensu stricto, B. japonica, B. garinii, B. sinica, and B. valaisiana. Significant differences in prevalence rates of BBSL were observed by species, landscape types, altitude, and season. Our findings indicate a wide distribution of multiple endemic BBSL genospecies based on a large-scale survey within Yunnan, which underline the need to expand surveillance efforts for human in southwestern China.

Description of Haemaphysalis (Alloceraea) Kolonini sp. nov., a new species in subgenus Alloceraea Schulze (Ixodidae: Haemaphysalis) in China.

Southeastern Asia has been postulated as probably the original sites of genus Haemaphysalis, where numerous structurally advanced haemaphysalids now occur. Because of its phylogenetic and biological interest, we report the discovery of a structurally primitive new species Haemaphysalis (A.) kolonini belonging to subgenus Alloceraea from Baoshan and Weixi cities, Yunnan province, southwestern China. Both the morphological and phylogenic characteristics of the species support congruently to erect Hae. (A.) kolonini as a new species. The male and female adults of Haemaphysalis (A.) kolonini are respectively described and illustrated. Taxonomic discussion and keys to Alloceraea species in China are also provided to be able to distinguish species that are close morphologically.

Investigation on Ehrlichia Infection in Small Mammals and Ticks from Tengchong, Yunnan Province, Southern China.

BACKGROUND: Rare investigation on tick-borne pathogens was carried out in Yunnan, China. In this study, we did a survey on Ehrlichia infection in small mammals and ticks. A total of 40 small mammals and 49 ticks were collected from Tengchong, Yunnan province. PCR targeting 16S ribosomal RNA (rRNA), citrate synthase, GroEL heat-shock protein operon, and major outer membrane protein genes was performed and positive amplicons were sequenced. RESULTS: The 40 small mammals were identified as 10 species, 2 (5.0%) of which were infected with Ehrlichia, 4 (10.0%) were infected with Anaplasma phagocytophilum and another 2 (5.0%) were infected with Candidatus Neoehrlichia mikurensis. Six (12.2%) ticks were positive for Ehrlichia and another two (4.1%) were infected with A. phagocytophilum. Neither small mammals nor ticks had coinfection. The detected Ehrlichia was named as Ehrlichia sp. YN04, which was in the same clade of Ehrlichia sp. 360 by phylogenetic analysis. The sequences of the pathogen recovered from small mammals and ticks were identical with each other. CONCLUSION: The study reports one Ehrlichia species first detected from small mammals and ticks in mainland China. As Yunnan is a famous "Global Biodiversity Hotspot" in the world, we may expect much more tick-borne infectious pathogens existing and declare more public health attention in this region.

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.

[Current situation and effect of non-hazardous treatment of excrement of sanitary latrines in rural schistosomiasis endemic areas of Eryuan County, Yunnan Province].

OBJECTIVE: To understand the condition of the construction and management of sanitary latrines, and assess the effect of disposal of the excrement in rural schistosomiasis endemic areas in Yunnan Province. METHODS: Three villages with schistosomiasis endemic were selected from Eryuan County and 30 households per village were sampled randomly for the field survey. The stool samples were sampled and tested according to the national standard. RESULTS: Totally 90 latrines were surveyed. The popularity rates of sanitary latrines in Yongle, Qiandian and Xinzhuang villages were 83.19%, 83.12% and 81.63% respectively. In the 90 household latrines, only 32.22% located inside the courtyard, and 91.67% of sanitary latrines and 70.00% of non-sanitary latrines had integrated buildings. Maggots or pupae or adult flies were found in 33.33% of sanitary latrines and all of non-sanitary latrines with the average amounts of 1.05 and 3.40 per latrine respectively. The removal rate of fecal coliform, the sedimentation rate of parasitic eggs and the mortality rate of Ascaris eggs were 90.00%, 80.61% and 95.20% on average respectively. The qualified rate of the fecal coliform of the outlet of the sanitary latrines was 41.67%, and the qualified rate of the mortality rate of Ascaris eggs was 78.13%. No living schistosome eggs were found at the outlets of latrines. For the effect of non-hazardous treatment, there was a statistically significant difference between the sanitary latrines and non-sanitary latrines. CONCLUSIONS: The latrine improvement has a good effect on non-hazardous treatment of the excrement in Yunnan Province, but the construction, application and management of sanitary latrines still need to be strengthened.

[Analysis of schistosomiasis endemic situation in Yunnan Province from 2009 to 2012].

OBJECTIVE: To evaluate the effect of schistosomiasis control in Yunnan Province since achieving the transmission control standards, so as to provide the evidence for formulating the next prevention and control strategy. METHODS: The schistosomiasis epidemic monitoring reports, annual reports, relevant information about Oncomrnelania hupensis snails, and schistosomiasis patient conditions were collected and analyzed for epidemic condition and characteristics of schistosomiasis in Yunnan Province from 2009 to 2012. RESULTS: The various epidemic monitoring indicators all decreased. In 2012, compared with 2009, the prevalence reduced by 33.33% after correction; the number of villages with schistosomiasis patients reduced by 55.56%; the adjusted positive rate of livestock stool tests reduced by 45.45%; the number of villages with infected cow reduced by 42.25%; the actual area with snails reduced by 13.58%; the density of living snails reduced by 25.66%, and no schistosome infected snails were found in 2012. CONCLUSION: There still exist schistosome infections in human and animals in some local areas of Yunnan Province, and it is difficult to achieve the standard of schistosomiasis transmission interrupted in a short period.

[Investigation on infection sources of schistosomiasis in mountainous regions, Nanjian County, Yunnan Province].

OBJECTIVE: To understand the types of schisotsome infection sources and their roles in schistosomiasis transmission in mountainous endemic regions in Yunnan Province, so as to provide the evidence for implementing the comprehensive control measures based on infection source control. METHODS: Two villages of typical mountainous regions in Nanjian County were chosen for field investigation. The Oncomelania hupensis snail status was surveyed by the methods of systematic and environmental sampling. The infections of schistosomiasis were surveyed in residents with the indirect haemagglutination and the hatching method, in livestock with the hatching method and in wild animals with the anatomical method and the hatching method. The distribution and pollution status of wild faeces were investigated in the snail environments nearby villages or with livestock frequent activities. The positives of schistosomiasis in wild faeces were tested with the hatching method. The pollution index of wild feces was calculated. RESULTS: A total of 533.56 hm2 were investigated in two villages, and the area with snails was 16.52 hm2. The rate of frame with snails was 1.03%, the average density of snails was 0.07 snails/0.1 m2, and no positive snails were found. The positive rate of blood examinations of population was 1.61%, but no persons were positive with the hatching method. The infection rates were 0.90%, and 0.62% in cattle and equus, respectively, and there were no positives in other livestock. Totally 472 piles of wild feces of 6 species (human, cattle, equus, goat, pig and dog) distributed in the investigation areas, and among them, the densities of wild feces of cattle, equus, and dog were 7.2, 4.3 piles/hm2, and 2.1 piles/hm2 respectively, being relatively higher than others. The hatching positive rates of wild faeces of cattle, equus, and dog were 2.27% (3/132), 2.63% (2/76), and 3.70% (1/27), respectively. The relative pollution indexes of wild faeces of cattle, equus, and dog were 80.68%, 15.89%, and 3.43%, respectively. CONCLUSIONS: After schistosomiasis transmission is controlled, the potential epidemic factors still remain in the mountainous endemic regions of schistosomiasis in Yunnan Province. The cattle are still the most infection source of schistosomiasis, but equus and dog as infection sources should be not neglected. We should extend the investigation and monitor scope of the infection sources, and carry out scientific and feasible control technique and management measures.

[Effects of three kinds of molluscicides in fields of mountainous areas of Yunnan Province].

The molluscicidal effects of 10% salt of quinoid- 2' ,5-dichloro-4'-nitrosalicylanilide (LDS) with dusting method, 26% amine molluscicide (MNSC) and 50% niclosamide (WPN) with insufflation method were tested in the fields of mountainous areas of Yunnan Province. After 7, 15 and 30 days of the tests, the death rates of snails, decline rates of living snail densities, and occurrence rates of frames with living snails of the 10% LDS group were 72.69%-87.16%, 74.10%-88.84%, and 20.00%-30.00%, respectively; those of the 26% MNSC group were 66.21%-91.06%, 69.55%-91.36%, and 19.44%-27.78% respectively; those of the 50% WPN group were 78.10%-90.18%, 82.44%-91.60%, 17.50%-20.00%, respectively. The results showed that the molluscicidal effects of 10% LDS, 26% MNSC and 50% WPN were basically equal.

[Molluscicidal effect of suspension concentrate of metaldehyde and niclosamide in fields of mountainous areas in Yunnan Province].

OBJECTIVE: To evaluate the molluscicidal effect of 26% suspension concentrate of metaldehyde and niclosamide (MNSC) in the fields of mountainous areas in Yunnan Province. METHODS: Some representative Oncomelania snail environments of schistosomiasis endemic regions were selected as study areas in Heqing County, Yunnan Province and 26% MNSC and 50% wettable powder of niclosamide ethanolamine salt (WPN) were used to kill the snails with the mechanical spraying and sprinkler spraying methods respectively. RESULTS: Seven, 15, 30 days after the spraying with the mechanical spraying method, in corn fields, the snail mortality rates were from 88.68% to 92.88% in the MNSC group, and from 89.86% to 90.32% in the WPN group respectively, and in the ditch, the snail mortality rates were from 89.97% to 94.46% in the MNSC group and from 90.08% to 96.74% in the WPN group respectively, and there was no significant difference between the 2 groups (P > 0.05); 7, 15, 30 days after the spraying with the sprinkler spraying method, in corn fields, the snail mortality rates were from 66.14% to 89.11% in the MNSC group, and from 78.40% to 91.22% in the WPN group, respectively, in the ditch, the snail mortality rates were from 84.13% to 94.27% in the MNSC group, and from 85.81% to 95.26% in the WPN group respectively, and there was no significant difference between the 2 groups (P > 0.05). CONCLUSION: 26% MNSC has good molluscicidal effect in the fields of mountainous areas in Yunnan Province

[Temporal trend and spatial distribution of premature mortality burden due to chronic obstructive pulmonary disease in urban Kunming for the years 1998-2003].

OBJECTIVE: To study the temporal trend and spatial distribution of premature mortality burden due to chronic obstructive pulmonary disease (COPD) in terms of years of life lost (YLL) in an urban region of Kunming for the years 1998-2003. METHODS: Pan Long district was selected as the study region. YLL was used to measure premature mortality burden due to COPD. The tate of YLL per 1000 residents was calculated without age-weighting but with 3% discounting rate and broken down by year. Geographic information system (GIS) technique was used to display the spatial distribution of premature mortality burden due to COPD during the six years of study. RESULTS: During the years from 1998 to 2003, the premature mortality burden due to COPD decreased by years while males seemed to have a higher YLL rate of COPD than females. Central areas remained to have higher YLL/1000 population values of COPD for the six years, whereas remote areas tended to have a decreasing trend of YLL/1000 population values of COPD in the same period. CONCLUSION: Central areas of the city should be emphasized in further control of COPD in the urban region of Kunming.