Epidemiological and clinical characteristics of scrub typhus in Guizhou Province, China: An outbreak study of scrub typhus.

The reported cases of scrub typhus (ST) have continued to escalate, with outbreaks occurring regionally in China. These pose an increasing public health threat at a time when public health has been overwhelmed. During the period from July to August 2022, in Rongjiang County, Guizhou Province, China, 13 out of 21 fever patients were diagnosed with scrub typhus, based on epidemiological investigation and blood test analysis. The major clinical symptoms of these patients showed fever, chills, headache, eschar, fatigue and pneumonia, which were accompanied by a rise in C-reactive protein, neutrophils, alanine transaminase (ALT) and aspartate aminotransferase (AST). Furthermore, nearly half of them exhibited abnormal electrocardiogram activity. Through semi-nested PCR, Sanger sequencing and phylogenetic tree construction, the Karp strain of Orientia tsutsugamushi (O. tsutsugamushi) was confirmed as the pathogen causing ST in Rongjiang County, which shared the same evolutionary branch with O. tsutsugamushi isolated from wild mouse liver or spleen, indicating that the wild mouse plays an important role in transmitting the disease. In contrast to the sporadic cases in the past, our study is the first to disclose an epidemic and the corresponding clinical characteristics of ST in Guizhou province, which is of great significance for the prevention and treatment of regional illnesses.

Changing epidemiologic patterns of typhus group rickettsiosis and scrub typhus in China, 1950-2022.

OBJECTIVES: We conducted a systematic analysis of the notifiable rickettsial diseases in humans in China during 1950-2022. METHODS: We utilized descriptive statistics to analyze the epidemiological characteristics, clinical manifestations, and diagnostic characteristics of typhus group rickettsiosis (TGR) and scrub typhus (ST) cases. RESULTS: Since the 1950s, there have been variations in the incidence rate of TGR and ST in China, with a downtrend for TGR and an uptrend for ST. The South became a high-incidence area of TGR, whereas the North was previously the high-incidence area. ST cases were concentrated in the South and the geographic area of ST spread northward and westward. The seasonality of TGR and ST were similar in the South but distinct in the North. Most TGR and ST cases were reported by county-level medical institutions, whereas primary institutions reported the least. Delayed diagnosis was associated with fatal outcomes of TGR and ST. Cases in low-incidence provinces, confirmed by laboratory tests and reported from county/municipal-level institutions had higher odds of delayed diagnoses. CONCLUSIONS: Our study revealed significant changes in the epidemiological characteristics of TGR and ST in China, which can provide useful information to enhance the control and prevention strategies of rickettsial diseases in China.

Molecular detection reveals diverse tick-borne bacterial and protozoan pathogens in two tick species from Yingshan County of Hubei Province, China in 2021-2022.

In this study, a total of 179 ticks infesting ruminant livestock, including 166 Haemaphysalis longicornis ticks and 13 Rhipicephalus microplus ticks were collected from Yingshan county of Hubei province, China in 2021-2022. PCR testing and sequence analysis revealed that the ticks infected with various species of pathogens including Rickettsia (R. japonica), Anaplasma (A. bovis, A. ovis, A. platys, and Ca. A. boleense), Ehrlichia (E. minasensis and Ehrlichia sp.), Theileria (T. orientalis and T. luwenshuni), and Babesia (B. bigemina). The infection rates of these pathogens were 0.56, 16.76, 7.26, 2.79 and 0.56%. respectively, while only 3 of 13 R. microplus ticks were detected to be infected wth Ehrlichia sp., A. bove., or T. luwenshuni. Our results revealed that a variety of tick-borne pathogens highly carried by these ticks, specially Ha. longicornis. Therefore, it is necessary to make effective control of the ticks and the tick-borne diseases in the County.

A seminested recombinase polymerase amplification assay to detect rickettsial pathogens in clinical samples.

Treatment at the early stage of onset is vital for the prognosis of rickettsioses. But the absence of specific clinical symptoms complicates the diagnosis of this condition. Herein we established a seminested recombinase polymerase amplification assay (snRPA-nfo) that enables quick detection and differentiation of rickettsial pathogens in clinical samples with high sensitivity and specificity. The conserved 17-kDa protein gene of Rickettsia sibirica and the 47-kDa protein gene of Orientia tsutsugamushi were targeted for the duplex RPA-nfo assay. The snRPA-nfo assay exhibited an increased LOD in spiked blood samples, up to 1000-fold in comparison to standard RPA-nfo, and a better detection rate (83.3%, 5/6) than TaqMan PCR (16.6%, 1/6, Ct ≤ 35) in clinically confirmed patient blood samples. Thus, snRPA-nfo assay represents a promising alternative to TaqMan PCR in the early diagnosis of rickettsioses for point-of-care testing as well as in resource-limited settings.

A liquid chromatography-miniature mass spectrometry (LC-Mini MS) method for quantitative analysis of risperidone and 9-hydroxyrisperidone in plasma.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a universal method for the quantitative analysis of small molecular drugs in therapeutic drug monitoring (TDM). Alternatively, liquid chromatography-miniature mass spectrometry (LC-Mini MS) is a simple operating technique for quantitative analysis. However, the wide chromatographic peaks and long retention times of TDM samples using the LC-Mini MS system deteriorated the accuracy and efficiency of quantitative analysis. Here, an optimized electrospray ionization (ESI) interface setup with a splitter valve and a capillary needle (I.D. 30 µm and O.D. 150 µm) of the LC-Mini MS system was acquired. The chromatographic peaks were narrower and smoother and the retention time was shorter for TDM compounds. Furthermore, a quantitative analysis method for risperidone and the active metabolite 9-hydroxyrisperidone in plasma was developed based on this optimal LC-Mini MS setup. The results showed that the calibration curves of risperidone and 9-hydroxyrisperidone had good linear ranges of 2-100 ng mL-1 (R2 = 0.9931) and 2-100 ng mL-1 (R2 = 0.9915), respectively. Finally, the matrix effects, recoveries and stability of risperidone and 9-hydroxyrisperidone samples were analyzed. The results satisfied the requirements of quantitative validation in routine TDM procedures.

Molecular Detection of Tick-Borne Bacterial and Protozoan Pathogens in Haemaphysalis longicornis (Acari: Ixodidae) Ticks from Free-Ranging Domestic Sheep in Hebei Province, China.

Ticks and tick-borne pathogens significantly threaten human and animal health worldwide. Haemaphysalis longicornis is one of the dominant tick species in East Asia, including China. In the present study, 646 Ha. longicornis ticks were collected from free-ranging domestic sheep in the southern region of Hebei Province, China. Tick-borne pathogens of zoonotic and veterinary importance (i.e., Rickettsia, Anaplasma, Ehrlichia, Borrelia, Theileria, and Hepatozoon spp.) were detected in the ticks using PCR assays and sequence analysis. The prevalence rates of these pathogens were 5.1% (33/646), 15.9% (103/646), 1.2% (8/646), 17.0% (110/646), 0.15% (1/646), and 0.15% (1/646), respectively. For Rickettsia spp., R. japonica (n = 13), R. raoultii (n = 6), and Candidatus R. jingxinensis (n = 14) were detected for the first time in the province, while several Anaplasma spp. were also detected in the ticks, including A. bovis (n = 52), A. ovis (n = 31), A. phagocytophilum (n = 10), and A. capra (n = 10). A putative novel Ehrlichia spp. was also found with a prevalence of 1.2% in the area. The present study provides important data for effectively controlling ticks and tick-borne diseases in the Hebei Province region of China.

Emergence of ehrlichiosis by a new tick-borne Ehrlichia species in China.

OBJECTIVES: From March to June 2021, the reported number of clinically diagnosed endemic typhus in Anhui and Hubei provinces of China nearly increased four-fold compared with the monthly average numbers in last 5 years. An etiological and epidemiological investigation was initiated. METHODS: The clinical specimens from the reported patients and the potential vector ticks were collected for molecular and serological detection, as well as cell culturing assay to identify the potential pathogen. RESULTS: Polymerase chain reaction and sequence analysis of rrs and groEL showed that the pathogen from these patients was Ehrlichia sp., isolated from Haemaphysalis longicornis attached to these patients. The phylogenetic analysis based on 39 Ehrlichia genomes suggested that it should be taxonomically classified as a novel species, tentatively named "Candidatus Ehrlichia erythraense". A total of 19 of 106 cases were confirmed as Candidatus Ehrlichia erythraense infections by polymerase chain reaction, sequencing, and/or serological tests. The most frequent symptoms were fever (100%), rashes (100%), asthenia (100%), anorexia (100%), and myalgia (79%). CONCLUSION: The occurrence of the disease presenting with fever and rashes in Anhui and Hubei provinces was caused by a novel species of the genus Ehrlichia; physicians need to be aware of this newly-discovered pathogen to ensure appropriate testing, treatment, and regional surveillance.

Clinical Forms of Japanese Spotted Fever from Case-Series Study, Zigui County, Hubei Province, China, 2021.

We report a case-series study of 5 patients with Japanese spotted fever from the Three Gorges Area in China, including 1 fatal case. Seroprevalence of Rickettsia japonica was ≈21% among the local population. Our report highlights the emerging potential threat to human health of Japanese spotted fever in the area.

Molecular detection of Rickettsiales and a potential novel Ehrlichia species closely related to Ehrlichia chaffeensis in ticks (Acari: Ixodidae) from Shaanxi Province, China, in 2022 to 2023.

Important tick-borne diseases include spotted fever group Rickettsia (SFGR), Anaplasma, and Ehrlichia, which cause harm to animal and human health. Ixodidae are the primary vectors of these pathogens. We aimed to analyze the prevalence and genetic diversity of SFGR, Anaplasma, and Ehrlichia species in the Ixodidae in Shaanxi Province, China. Herein, 1,113 adult Ixodidae ticks were collected from domestic cattle and goats, and detected using nested PCR. A total of four Ixodidae species were collected and Ca. R. jingxinensis (20.58%, 229/1113), A. bovis (3.05%, 34/1113), A. capra (3.32%, 37/1113), A. marginale (0.18%, 2/1113), E. sp. Yonaguni138 (0.18%, 2/1113), and a potent novel Ehrlichia species named E. sp. Baoji96 (0.09%, 1/1113) were detected. A. marginale was detected for the first time in Rhipicephalus microplus. E. sp. Baoji96 was closely related to E. chaffeensis and was first identified in Haemaphysalis longicornis. In addition, co-infection with two Rickettsiales pathogens within an individual tick was detected in 10 (1.54%) ticks. This study provides a reference for the formulation of biological control strategies for ticks and tick-borne diseases in Shaanxi Province, and could lead to an improved control effect.

Human Rickettsia felis infections in Mainland China.

We identified four flea-borne spotted fever cases caused by Rickettsia felis in a retrospective survey of 182 patients with fever of unknown origin (FUO) in China between 2021 and 2022. The clinical signs and symptoms of the patients were similar to those of other rickettsioses, including fever, rash, and liver and kidney dysfunction. All four patients in the present study developed pneumonia or lung lesions after R. felis infection. The cases of R. felis infection, a neglected infectious disease, were sporadic in multiple provinces of the country. The high prevalence (2.14%, 4/187) of R. felis among patients with FUO highlights the risk posed by this pathogen to public health in China.

Anaplasma bovis Infection in Fever and Thrombocytopenia Patients - Anhui Province, China, 2021.

What is already known about this topic?: The genusAnaplasma contains seven recognized bacterial species, mainly transmitted by tick bites. The two species, A. phagocytophilum and A. capra, are known commonly to cause diseases in humans. What is added by this report?: Anaplasma bovis was initially thought to be only an animal agent until the first patient case was reported in 2019. This study investigated another two patients who became sick within one month in the same township and were infected with A. bovis in Anhui Province. What are the implications for public health practice?: This study suggested that more A. bovis-infected patients may exist in this area and that patients with anaplasmosis require an early and specific diagnosis.

Severe Case of Rickettsiosis Identified by Metagenomic Sequencing, China.

A case of Rickettsia sibirica subspecies sibirica BJ-90 infection in China was identified by metagenomic analysis of an eschar biopsy specimen and confirmed by nested PCR. Seroprevalence of spotted fever group Rickettsia was ≈17.4% among the local population. This report highlights the threat of rickettsioses to public health in the Qinghai-Tibet Plateau.

Beyond a Ribosomal RNA Methyltransferase, the Wider Role of MraW in DNA Methylation, Motility and Colonization in Escherichia coli O157:H7.

MraW is a 16S rRNA methyltransferase and plays a role in the fine-tuning of the ribosomal decoding center. It was recently found to contribute to the virulence of Staphylococcus aureus. In this study, we examined the function of MraW in Escherichia coli O157:H7 and found that the deletion of mraW led to decreased motility, flagellar production and DNA methylation. Whole-genome bisulfite sequencing showed a genome wide decrease of methylation of 336 genes and 219 promoters in the mraW mutant including flagellar genes. The methylation level of flagellar genes was confirmed by bisulfite PCR sequencing. Quantitative reverse transcription PCR results indicated that the transcription of these genes was also affected. MraW was furtherly observed to directly bind to the four flagellar gene sequences by electrophoretic mobility shift assay (EMSA). A common flexible motif in differentially methylated regions (DMRs) of promoters and coding regions of the four flagellar genes was identified. Reduced methylation was correlated with altered expression of 21 of the 24 genes tested. DNA methylation activity of MraW was confirmed by DNA methyltransferase activity assay in vitro and repressed by DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza). In addition, the mraW mutant colonized poorer than wild type in mice. We also found that the expression of mraZ in the mraW mutant was increased confirming the antagonistic effect of mraW on mraZ. In conclusion, mraW was found to be a DNA methylase and have a wide-ranging effect on E. coli O157:H7 including motility and virulence in vivo via genome wide methylation and mraZ antagonism.

An integrated strategy to identify genes responsible for sesquiterpene biosynthesis in turmeric.

KEY MESSAGE: Metabolic module, gene expression pattern and PLS modeling were integrated to precisely identify the terpene synthase responsible for sesquiterpene formation. Functional characterization confirmed the feasibility and sensitivity of this strategy. Plant secondary metabolite biosynthetic pathway elucidation is crucial for the production of these compounds with metabolic engineering. In this study, an integrated strategy was employed to predict the gene function of sesquiterpene synthase (STS) genes using turmeric as a model. Parallel analysis of gene expression patterns and metabolite modules narrowed the candidates into an STS group in which the STSs showed a similar expression pattern. The projections to latent structures by means of partial least squares model was further employed to establish a clear relationship between the candidate STS genes and metabolites and to predict three STSs (ClTPS16, ClTPS15 and ClTPS14) involved in the biosynthesis of several sesquiterpene skeletons. Functional characterization revealed that zingiberene and ß-sesquiphellandrene were the major products of ClTPS16, and ß-eudesmol was produced by ClTPS15, both of which indicated the accuracy of the prediction. Functional characterization of a control STS, ClTPS1, produced a small amount of ß-sesquiphellandrene, as predicted, which confirmed the sensitivity of metabolite module analysis. This integrated strategy provides a methodology for gene function predictions, which represents a substantial improvement in the elucidation of biosynthetic pathways in nonmodel plants.

[Comparison of volatile components in Schisandra propinqua from different habitats by HS-SPME-GC-MS].

A headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) method was developed and optimized for qualitative and semi quantitative analysis of volatile components from Schisandra propinqua, a kind of Yi Nationality herb medicine. This method was used for analysis and evaluation of volatile components from S.propinqua from four different geometrical origins (Yunnan Wuding, Yunnan Luoping, Guizhou Qingzhen and Hubei Shennongjia). 51, 53, 52， 50 compounds were identified from the above four kinds of samples respectively, in which 46 volatile components were contained in all of these four origins. The volatile components in these samples from different geographical origins were then compared by using principal component analysis, cluster analysis and other chemometrics methods. The results suggested that the analysis of volatile components can be used to distinguish the S.propinqua from four origins, and those samples from close geographical origins were more similar in volatile components. The developed method was stable, reliable, and suitable for rapid analysis of volatile components from S.propinqua, providing reference for quality control, drug development and scientific utilization of the herb.

Construction and Quality Analysis of Transgenic Rehmannia glutinosa Containing TMV and CMV Coat Protein.

Plant viruses, especially tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) are serious threats to Rehmannia glutinosa which is a "top grade" herb in China. In the present study, TMV- and CMV-resistant Rehmannia glutinosa Libosch. plants were constructed by transforming the protein (CP) genes of TMV and CMV into Rehmannia glutinosa via a modified procedure of Agrobacterium tumefaciens-mediated transformation. Integration and expression of TMV CP and CMV CP transgenes in 2 lines, LBA-1 and LBA-2, were confirmed by PCR, Southern blot and RT-PCR. Both LBA-1 and LBA-2 were resistant to infection of homologous TMV and CMV strains. The quality of transgenic Rehmanniae Radix was evaluated based on fingerprint analysis and components quantitative analysis comparing with control root tubes. These results showed that chemical composition of transgenic Rehmanniae Radix were similar to non-transgenic ones, which demonstrated that the medical quality and biosafety of transgenic Rehmanniae Radix were equivalent to non-transgenic material when consumed as traditional Chinese medicinal (TCM).

Functional characterization of ent-copalyl diphosphate synthase, kaurene synthase and kaurene oxidase in the Salvia miltiorrhiza gibberellin biosynthetic pathway.

Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes. Its bioactive diterpenoid tanshinones have been reported to have many pharmaceutical activities, including antibacterial, anti-inflammatory, and anticancer properties. Previous studies found four different diterpenoid biosynthetic pathways from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) in S. miltiorrhiza. Here, we describe the functional characterization of ent-copalyl diphosphate synthase (SmCPSent), kaurene synthase (SmKS) and kaurene oxidase (SmKO) in the gibberellin (GA) biosynthetic pathway. SmCPSent catalyzes the cyclization of GGPP to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by SmKS. Then, SmKO catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid. Our results show that the fused enzyme SmKS-SmCPSent increases ent-kaurene production by several fold compared with separate expression of SmCPSent and SmKS in yeast strains. In this study, we clarify the GA biosynthetic pathway from GGPP to ent-kaurenoic acid and provide a foundation for further characterization of the subsequent enzymes involved in this pathway. These insights may allow for better growth and the improved accumulation of bioactive tanshinones in S. miltiorrhiza through the regulation of the expression of these genes during developmental processes.

[Research progress of genetic engineering on medicinal plants].

The application of genetic engineering technology in modern agriculture shows its outstanding role in dealing with food shortage. Traditional medicinal plant cultivation and collection have also faced with challenges, such as lack of resources, deterioration of environment, germplasm of recession and a series of problems. Genetic engineering can be used to improve the disease resistance, insect resistance, herbicides resistant ability of medicinal plant, also can improve the medicinal plant yield and increase the content of active substances in medicinal plants. Thus, the potent biotechnology can play an important role in protection and large area planting of medicinal plants. In the development of medicinal plant genetic engineering, the safety of transgenic medicinal plants should also be paid attention to. A set of scientific safety evaluation and judgment standard which is suitable for transgenic medicinal plants should be established based on the recognition of the particularity of medicinal plants.

A comparative study on volatile metabolites profile of Dracaena cochinchinensis (Lour.) S.C. Chen xylem with and without resin using GC-MS.

Dragon's blood is a famous traditional Chinese medicine produced from source plants under bio- or abio-stress. Dracaena cochinchinensis (Lour.) S.C. Chen xylem (DX) is one of the most important sources of the medicine. In this work, a GC-MS method was developed for analysis of the n-hexane extracts of DX with resin (DXR) and without resin (DXW). The repeatability of the method was also investigated for a metabolite comparative study of the different xylems. About 80 components were detected, 26 of which were identified in both DXR and DXN. Three sesquiterpenes (τ-cadinol, τ-muurolon and α-cadinol) were first discovered in Dracaena cochinchinensis (Lour.) S.C. Chen. The chromatographs of the two plant materials were compared and differences of compounds were found. It showed that phytosterols showed a dramatic rise in content, and sesquiterpenes were found to be synthesized in DXR.