Co-Localization of Sampling and Sequencing for Zoonotic Pathogen Identification in the Field Monitoring Using Mobile Laboratories.

Introduction: Accurate etiological detection is needed to evaluate the risk of zoonotic diseases. Metagenomic next-generation sequencing (mNGS) can be used to monitor pathogens in animal species and identify potential zoonotic threats. The current sampling model for zoonotic pathogen monitoring in wild animals requires samples to be transferred from the field to a laboratory for further detection. Methods: We constructed a zoonotic pathogen survey model using a set of mobile laboratories. Results: The monitoring in this study was preplanned to detect Yersinia pestis, but the mNGS unexpectedly identified Bartonella spp. in the rodent samples, thus exposing the threat of bartonellosis to humans in this region. The co-localization of sampling and sequencing (CLOSS) model we tested required no long-distance transferring of samples and expands the regional coverage of zoonotic surveys by using a mobile laboratory. Discussion: Using this mNGS technique will enable detection of more zoonotic pathogens beyond the preplanned monitoring targets. This may increase the surveillance efficiency compared with that of the previous workflow and expand the application of the mobile laboratories for infectious diseases identification and surveillance in the field.

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.

Yersinia pestis Interacts With SIGNR1 (CD209b) for Promoting Host Dissemination and Infection.

Yersinia pestis, a Gram-negative bacterium and the etiologic agent of plague, has evolved from Yersinia pseudotuberculosis, a cause of a mild enteric disease. However, the molecular and biological mechanisms of how Y. pseudotuberculosis evolved to such a remarkably virulent pathogen, Y. pestis, are not clear. The ability to initiate a rapid bacterial dissemination is a characteristic hallmark of Y. pestis infection. A distinguishing characteristic between the two Yersinia species is that Y. pseudotuberculosis strains possess an O-antigen of lipopolysaccharide (LPS) while Y. pestis has lost the O-antigen during evolution and therefore exposes its core LPS. In this study, we showed that Y. pestis utilizes its core LPS to interact with SIGNR1 (CD209b), a C-type lectin receptor on antigen presenting cells (APCs), leading to bacterial dissemination to lymph nodes, spleen and liver, and the initiation of a systemic infection. We therefore propose that the loss of O-antigen represents a critical step in the evolution of Y. pseudotuberculosis into Y. pestis in terms of hijacking APCs, promoting bacterial dissemination and causing the plague.

Molecular Evidence of Human Monkeypox Virus Infection, Sierra Leone.

Monkeypox virus is a zoonotic disease endemic to Africa. In 2017, we confirmed a case of human monkeypox virus in Sierra Leone by molecular and serologic methods. Sequencing analysis indicated the virus belongs to the West African clade and data suggest it was likely transmitted by wild animals.

Endemic Melioidosis in Southern China: Past and Present.

Melioidosis is a severe tropical infectious disease caused by the soil-dwelling bacterium Burkholderia pseudomallei, predominantly endemic to Southeast Asia and northern Australia. Between the 1970s and the 1990s, the presence of B. pseudomallei causing melioidosis in humans and other animals was demonstrated in four coastal provinces in southern China: Hainan, Guangdong, Guangxi, and Fujian, although indigenous cases were rare and the disease failed to raise concern amongst local and national health authorities. In recent years, there has been a rise in the number of melioidosis cases witnessed in the region, particularly in Hainan. Meanwhile, although China has established and maintained an effective communicable disease surveillance system, it has not yet been utilized for melioidosis. Thus, the overall incidence, social burden and epidemiological features of the disease in China remain unclear. In this context, we present a comprehensive overview of both historical and current information on melioidosis in Southern China, highlighting the re-emergence of the disease in Hainan. Surveillance and management strategies for melioidosis should be promoted in mainland China, and more research should be conducted to provide further insights into the present situation.

Yersinia pseudotuberculosis Exploits CD209 Receptors for Promoting Host Dissemination and Infection.

Yersinia pseudotuberculosis is a Gram-negative enteropathogen and causes gastrointestinal infections. It disseminates from gut to mesenteric lymph nodes (MLNs), spleen, and liver of infected humans and animals. Although the molecular mechanisms for dissemination and infection are unclear, many Gram-negative enteropathogens presumably invade the small intestine via Peyer's patches to initiate dissemination. In this study, we demonstrate that Y. pseudotuberculosis utilizes its lipopolysaccharide (LPS) core to interact with CD209 receptors, leading to invasion of human dendritic cells (DCs) and murine macrophages. These Y. pseudotuberculosis-CD209 interactions result in bacterial dissemination to MLNs, spleens, and livers of both wild-type and Peyer's patch-deficient mice. The blocking of the Y. pseudotuberculosis-CD209 interactions by expression of O-antigen and with oligosaccharides reduces infectivity. Based on the well-documented studies in which HIV-CD209 interaction leads to viral dissemination, we therefore propose an infection route for Y. pseudotuberculosis where this pathogen, after penetrating the intestinal mucosal membrane, hijacks the Y. pseudotuberculosis-CD209 interaction antigen-presenting cells to reach their target destinations, MLNs, spleens, and livers.

A Novel Francisella-Like Endosymbiont in Haemaphysalis longicornis and Hyalomma asiaticum, China.

Francisella tularensis causes a highly infectious zoonotic disease tularemia. Both Haemaphysalis longicornis and Hyalomma asiaticum are widely distributed in China, but the presence of Francisella and Francisella-like endosymbionts (FLEs) in the two tick species is poorly understood. Therefore, a total of 627 H. longicornis (471 adults and 156 nymphs) and 88 Hy. asiaticum ticks (adults) were collected, of which 88 were from Bole of Xinjiang, 236 from Liaoyang, and 176 from Shenyang of Liaoning, and 215 from Wuhan of Hubei. Notably, five H. longicornis pools from Liaoyang of Liaoning province might have harbored F. tularensis, showing a minimum prevalence of 2.12% (5/236). This study should alert the health department and veterinarians working within the region to prevent and control the emergence of tularemia. After the screening of 16S rRNA and tul4 genes, the results revealed that FLEs were detected in Hy. asiaticum ticks in Bole and in H. longicornis ticks in Liaoyang and Shenyang. Their infection rate was 100% (88/88), 3.39% (8/236 is a minimum), and 8.52% (15/176), respectively. Phylogenetic analyses indicated that the sequence named bole in Hy. Asiaticum from Bole, the sequence named liaoyang1 in H. longicornis from Liaoyang, and the sequence named shanyang1 in H. longicornis from Shenyang shared consistent 16S rRNA sequence, and the difference between Chinese FLEs and the known FLEs was obvious. These findings suggest that this FLE species might be a potentially novel FLE circulating in H. longicornis and Hy. asiaticum from China.

Reemergence of human plague in Yunnan, China in 2016.

The third plague pandemic originated from Yunnan Province, China in the middle of the 19th century. The last human plague epidemic in Yunnan occurred from 1986-2005. On June 6, 2016, a case of human plague was reported in the Xishuangbanna Prefecture, Yunnan. The patient suffered from primary septicemic plague after exposure to a dead house rat (Rattus flavipectus), which has been identified as the main plague reservoir in the local epizootic area. Moreover, a retrospective investigation identified another bubonic plague case in this area. Based on these data, human plague reemerged after a silent period of ten years. In this study, three molecular typing methods, including a clustered regularly interspaced short palindromic repeats (CRISPR) analysis, different region analysis (DFR), and multiple-locus variable number of tandem repeats analysis (MLVA), were used to illustrate the molecular characteristics of Yersinia pestis (Y. pestis) strains isolated in Yunnan. The DFR profiles of the strains isolated in Yunnan in 2016 were the same as the strains that had previously been isolated in this Rattus flavipectus plague focus. The c3 spacer present in the previously isolated strains was absent in the spacer arrays of the Ypc CRISPR loci of the strains isolated in 2016. The MLVA analysis using MLVA (14+12) showed that the strains isolated from the human plague case and host animal plague infection in 2016 in Yunnan displayed different molecular patterns than the strains that had previously been isolated from Yunnan and adjacent provinces.

A case of Francisella tularensis subspecies holarctica in China.

We report a highly unusual case of ulceroglandular tularemia in Beijing, China. The serological texting, and sequencing of three specific genes by PCR analysis, suggested that this case was infected by Francisella tularensis. Next, using 15 canonical single-nucleotide polymorphisms and insertion-deletion markers (SNPs-INDELs) and five variable-number tandem repeat loci (VNTRs), this case was assigned to a known clade from Russia, and not to the four clades that were previously identified, including previous Chinese isolates. The case that is reported herein provides evidence of type B tularemia in Beijing, and it demonstrates unprecedented levels of diversity of the Chinese variant of F. tularensis.

[Establishment and evaluation of identification method for Yersinia pestis and Yersinia pseudotuberculosis].

OBJECTIVE: To establish a gene identification method of Yersinia pestis and Yersinia pseudotuberculosis for plague surveillance. METHODS: According to the specific genomic sequences of Y. pestis and Y. pseudotuberculosis, i.e. "pestis Island (PeI)" and "pseudotuberculosis Island (PsI)" and the published genomic sequences of 12 strains of Y. pestis and 4 strains of Y. pseudotuberculosis, the specific identification primers of these sequences were designed. RESULTS: A total of 52 strains of Y. pestis and 57 strains of Y. pseudotuberculosis and other intestinal bacteria strains were tested with PCR. Of the 5 pairs of Y. pestis identification primers, PeI2 and PeI11 were specific for Y. pestis. Besides Y. pestis, the primers PeI1, PeI3 and PeI12 could detect part of 57 Y. pseudotuberculosis strains. Of the 5 pairs of Y. pseudotuberculosis identification primers, PsI1 could detect all the 52 strains of Y. pestis and 57 strains of Y. pseudotuberculosis. PsI7, PsI16, PsI18 and PsI19 were specific for Y. pseudotuberculosis. CONCLUSION: The primers PsI1, PeI 2 and PeI11, PsI7, PsI16, PsI18 and PsI19 can be used in the rapid identification of Y. pestis and Y. pseudotuberculosis, which can be also used to explore the circulation of atypical Y. pestis in quiescent plague foci.

Host Langerin (CD207) is a receptor for Yersinia pestis phagocytosis and promotes dissemination.

Yersinia pestis is a Gram-negative bacterium that causes plague. After Y. pestis overcomes the skin barrier, it encounters antigen-presenting cells (APCs), such as Langerhans and dendritic cells. They transport the bacteria from the skin to the lymph nodes. However, the molecular mechanisms involved in bacterial transmission are unclear. Langerhans cells (LCs) express Langerin (CD207), a calcium-dependent (C-type) lectin. Furthermore, Y. pestis possesses exposed core oligosaccharides. In this study, we show that Y. pestis invades LCs and Langerin-expressing transfectants. However, when the bacterial core oligosaccharides are shielded or truncated, Y. pestis propensity to invade Langerhans and Langerin-expressing cells decreases. Moreover, the interaction of Y. pestis with Langerin-expressing transfectants is inhibited by purified Langerin, a DC-SIGN (DC-specific intercellular adhesion molecule 3 grabbing nonintegrin)-like molecule, an anti-CD207 antibody, purified core oligosaccharides and several oligosaccharides. Furthermore, covering core oligosaccharides reduces the mortality associated with murine infection by adversely affecting the transmission of Y. pestis to lymph nodes. These results demonstrate that direct interaction of core oligosaccharides with Langerin facilitates the invasion of LCs by Y. pestis. Therefore, Langerin-mediated binding of Y. pestis to APCs may promote its dissemination and infection.

[Discovery and tracking source of the new subgroup of Chinese Francisella tularensis type B].

OBJECTIVE: To perform laboratory diagnosis and tracking source of a suspected tularemia patient in Beijing. METHODS: A suspected tularemia patient was reported in Beijing city on July 19, 2012. Genomic DNA was extracted from the blood sample of the patient, then general PCR and sequencing of amplicons were conducted using 3 specific genes (fopA, tul4 and 16S rRNA) Francisella tularensis (F.tularensis), and 2 genotyping primers (C1C4 and RD1). Two other laboratories repeated the PCR and sequencing of the fopA in parallel. At the same time, real-time PCR fluorescent ration was performed using 4 targets (fopA, ISFtul2, 23kDa, and tul4), and phylogenetic analysis was carried out using 11 canonical single nucleotide polymorphisms (SNPs) and 4 insertions or deletions. RESULTS: All the 3 specific genes were amplified positively, and sequenced fragments were 409, 407 and 1 053 bp, respectively. The patient was infected by F. tularensis comparing with the whole genome published. Next, amplicons of 151 and 924 bp were obtained by the 2 typing primers after sequencing, respectively. The segment lengths suggested that the patient was infected by the subsp. holarctica. All of the two other laboratories obtained positive data for the PCR and sequencing of the fopA. In addition, all the 4 targets tested positive by real-time PCR for F. tularensis. The Ct value of the fopA, ISFtul2, 23kDa and tul4 were 30, 25, 28, and 30, respectively. The phylogenetic analysis indicated that the whole genome of this case was assigned to a known clade from Russia, which was subgroup B3. CONCLUSION: This case was confirmed to be a tularemia patient, and a new subgroup of F. tularensis type B was found in China.

[Genetic diversity of Francisella tularensis subsp. holarctica in China].

OBJECTIVE: To explore the genetic relationship between the Chinese and the foreign species of Francisella tularensis. METHODS: Based on our own findings and from the literature, 17 SNP, 4 INDEL, and 12 VNTR were selected for phylogenetic analysis on 39 strains of F.tularensis, including 10 strains of Chinese F. tularensis and 29 strains of foreign F. tularensis that had been sequenced and published. SNP-INDEL and MLVA were used for the separation and combination. RESULTS: Data from the combined analysis indicated that 3 strains of Chinese F. tularensis with Japanese FSC022 were assigned to B5; 3 strains, with Swedish FSC200 to B1; 3 strains with American OSU18 to B2 and 1 strain with French FTNF002-00, German F92, and American OR96246 to B4, respectively. 10 strains of Chinese F. tularensis were assigned to 4 clades and the result demonstrated a wide diversity of F. tularensis subsp.holarctica in China. CONCLUSION: A set of simple and robust typing tools for F. tularensis subsp.holarctica were established in this study. Based on the results, F. tularensis subsp.holarctica might have had its origins in Asia.

Chromosomal rearrangement features of Yersinia pestis strains from natural plague foci in China.

The Yersinia pestis chromosome contains a large variety and number of insert sequences that have resulted in frequent chromosome rearrangement events. To identify the chromosomal rearrangement features of Y. pestis strains from five typical plague foci in China and study spontaneous DNA rearrangements potentially stabilized in certain lineages of Y. pestis genomes, we examined the linking mode of locally collinear blocks (LCBs) in 30 Y. pestis strains by a polymerase chain reaction-based method. Our results suggest most strains have relatively stable chromosomal arrangement patterns, and these rearrangement characteristics also have a very close relationship with the geographical origin. In addition, some LCB linking modes are only present in specific strains. We conclude Y. pestis chromosome rearrangement patterns may reflect the genetic features of specific geographical areas and can be applied to distinguish Y. pestis isolates; furthermore, most of the rearrangement events are stable in certain lineages of Y. pestis genomes.

Diversity of Francisella tularensis subsp. holarctica lineages, China.

We analyzed 10 isolates of Francisella tularensis subspecies holarctica from China and assigned them to known clades by using canonical single-nucleotide polymorphisms. We found 4 diverse subtypes, including 3 from the most basal lineage, biovar japonica. This result indicates unprecedented levels of diversity from a single region and suggests new models for emergence.

[Study on the spatial and temporal distribution of animal plague in Junggar Basin plague focus].

OBJECTIVE: To explore the spatial and temporal distributions of animal plague in Junggar Basin natural plague focus. METHODS: Data regarding plague antibody (F1) in serum of Great Gerbil (Rhombomys opimus, R. opimus) which were collected from 2005 to 2012 in Junggar Basin and analyzed. The changing rates on the positivity of F1 that appeared spatially and temporally were also analyzed. RESULTS: A total of 4 825 R. opimus serum samples were collected in 13 administrative regions in Junggar Basin. RESULTS: showed that plague R. opimus existed in two areas-Gurbantonggut desert in the eastern-center and the clay desert of western Junggar Basin. However, in these two areas, the intensity of animal plague prevalence was different. In the former region where Yesinia pestis positive serum was detected from R. opimus, the detected rate of R. opimus was 8.39%. However, in the latter areas, the average positive rate was 1.56%. The changing trends of R. opimus plague prevalence were also varied annually. In the western Junggar Basin, the trend showed a slowly downward profile. The serum positive rate of R. opimus for Yesinia pestis decreased, from 7.59% in 2005 to 0.61% in 2008, and appeared as a resting state that none of the positive sample could be found since then. However, in the eastern-center Junggar Basin area-also named as Gurbantonggut desert which had been divided into 3 segments(western, central and eastern, according to related geographical characteristics), the changing trends of animal plague seemed quite complex. In the western segment, the animal plague had two epidemic peaks-in 2006 and 2010, with the interval of 4 years, with the higher peak of all the three geographic segments as 45.65% in 2010 and the positive serum of R. opimus for plague could be detected each year from 2006 to 2012. However, there were 3 epidemic peaks in the same period in the central and eastern segments. In the central segment, the peaks appeared in 2006, 2009 and 2011, with the intervals as 2.5 years and the average positive rate 8.92% was seen the lowest in Gurbantonggut desert. In the eastern segment, the first 2 peaks appeared the same season as in the central segment, but the third peak appeared in 2012, with the peak interval as 3 years. The positive rate of R. opimus for plague was also different in seasons, with the positive rate higher in autumn than in spring. These findings showed that the animal plague could be continuously prevalent from spring to autumn in the natural foci of plague in the Junggar Basin. CONCLUSION: Both geographical and temporal fluctuations of animal plague existed in the natural foci of Junggar Basin which was also named as geographical heterogeneity. Consequently, animal plague could be divided into two areas-the clay plains desert in the western and the Gurbantonggut desert in the eastern-center Junggar Basin.

Genetic relationship between Francisella tularensis strains from China and from other countries.

OBJECTIVE: To study the types of subspecies of Francisella tularensis from China and to investigate the genetic relationships between F. tularensis strains from China and from other countries. METHODS: Ten strains of F. tularensis isolated from China were amplified by using typing primers C1/C4 and RD1. On the basis of the lengths of the polymerase chain reaction (PCR) products, it was concluded that these strains of F. tularensis belonged to the same subspecies. At the same time, the fopA, tul4, and 16S rRNA genes of the 10 strains were amplified, and a three-gene based phylogenetic analysis was performed using the Molecular Evolutionary Genetics Analysis software version 4.0. RESULTS: The 10 strains of F. tularensis from China were all identified as belonging to subspecies holarctica (type B). We found no direct relationship between the genotypes of F. tularensis subsp. holarctica and the geographical area from where they were isolated. CONCLUSION: The F. tularensis strains isolated from North China mainly belong to subspecies holarctica (type B). The strains of F. tularensis subsp. holarctica from China may have evolved earlier than those from Europe and North America.