Skin infectome of patients with a tick bite history.

Introduction: Ticks are the most important obligate blood-feeding vectors of human pathogens. With the advance of high-throughput sequencing, more and more bacterial community and virome in tick has been reported, which seems to pose a great threat to people. Methods: A total of 14 skin specimens collected from tick-bite patients with mild to severe symptoms were analyzed through meta-transcriptomic sequencings. Results: Four bacteria genera were both detected in the skins and ticks, including Pseudomonas, Acinetobacter, Corynebacterium and Propionibacterium, and three tick-associated viruses, Jingmen tick virus (JMTV), Bole tick virus 4 (BLTV4) and Deer tick mononegavirales-like virus (DTMV) were identified in the skin samples. Except of known pathogens such as pathogenic rickettsia, Coxiella burnetii and JMTV, we suggest Roseomonas cervicalis and BLTV4 as potential new agents amplified in the skins and then disseminated into the blood. As early as 1 day after a tick-bite, these pathogens can transmit to skins and at most four ones can co-infect in skins. Discussion: Advances in sequencing technologies have revealed that the diversity of tick microbiome and virome goes far beyond our previous understanding. This report not only identifies three new potential pathogens in humans but also shows that the skin barrier is vital in preventing horizontal transmissions of tick-associated bacteria or virus communities to the host. It is the first research on patients' skin infectome after a tick bite and demonstrates that more attention should be paid to the cutaneous response to prevent tick-borne illness.

Virome and Blood Meal-Associated Host Responses in Ixodes persulcatus Naturally Fed on Patients.

The long-lasting co-evolution of ticks with pathogens results in mutual adaptation. Blood-feeding is one of the critical physiological behaviors that have been associated with the tick microbiome; however, most knowledge was gained through the study of laboratory-reared ticks. Here we detached Ixodes persulcatus ticks at different stages of blood-feeding from human patients and performed high-throughput transcriptomic analysis on them to identify their virome and genes differentially expressed between flat and fully fed ticks. We also traced bloodmeal sources of those ticks and identified bats and three other potential mammalian hosts, highlighting the public health significance. We found Jingmen tick virus and 13 putative new viruses belonging to 11 viral families, three of which even exhibited high genetic divergence from viruses previously reported in the same tick species from the same geographic region. Furthermore, differential expression analysis suggested a downregulation of antioxidant genes in the fully fed I. persulcatus ticks, which might be related to bloodmeal-related redox homeostasis. Our work highlights the significance of active surveillance of tick viromes and suggests a role of reactive oxygen species (ROS) in modulating changes in the microbiome during blood-feeding.

Cutaneous Immunoprofiles of Three Spotted Fever Group Rickettsia Cases.

Spotted fever group rickettsia (SFGR) can cause mild to fatal illness. The early interaction between the host and rickettsia in skin is largely unknown, and the pathogenesis of severe rickettsiosis remains an important topic. A surveillance of SFGR infection by PCR of blood and skin biopsy specimens followed by sequencing and immunohistochemical (IHC) detection was performed on patients with a recent tick bite between 2013 and 2016. Humoral and cutaneous immunoprofiles were evaluated in different SFGR cases by serum cytokine and chemokine detection, skin IHC staining, and transcriptome sequencing (RNA-seq). A total of 111 SFGR cases were identified, including 79 "Candidatus Rickettsia tarasevichiae," 22 Rickettsia raoultii, 8 Rickettsia sibirica, and 2 Rickettsia heilongjiangensis cases. The sensitivity to detect SFGR in skin biopsy specimens (9/24, 37.5%) was significantly higher than that in blood samples (105/2,671, 3.9%) (P < 0.05). As early as 1 day after the tick bite, rickettsiae could be detected in the skin. R. sibirica infection was more severe than "Ca Rickettsia" and R. raoultii infections. Increased levels of serum interleukin-18 (IL-18), IP10, and monokine induced by gamma interferon (MIG) and decreased levels of IL-2 were observed in febrile patients infected with R. sibirica compared to those infected with "Ca Rickettsia." RNA-seq and IHC staining could not discriminate between SFGR-infected and uninfected tick bite skin lesions. However, the type I interferon (IFN) response was differently expressed between R. sibirica and R. raoultii infections at the cutaneous interface. It is concluded that skin biopsy specimens were more reliable for the detection of SFGR infection in human patients although the immunoprofile may be complicated by immunomodulators induced by the tick bite.

Emergence of human infection with Jingmen tick virus in China: A retrospective study.

BACKGROUND: A tick-borne segmented RNA virus called Jingmen tick virus (JMTV) was recently identified, variants of which were detected in a non-human primate host and fatal patients with Crimean-Congo haemorrhagic fever. We investigated its infectivity and pathogenicity for humans. METHODS: We obtained skin-biopsy, blood and serum samples from patients with tick bites, and used high-throughput sequencing, in situ hybridisation, and serologic testing to diagnose and ascertain the cases of JMTV infection. FINDINGS: A JMTV strain was isolated from the tick Amblyomma javanense into an embryo-derived tick cell line. We obtained sustained passage of JMTV, and revealed that it was able to accumulate in salivary glands of experimentally infected ticks. Four JMTV-infected patients were identified by high-throughput sequencing of skin biopsies and blood samples. The virus replication in skin tissue was visualised by in situ hybridisation. The four patients all had an itchy or painful eschar at the site of tick bite, with or without lymphadenopathy. Immunohistochemical examination revealed remarkable local inflammation manifested as infiltration by neutrophils. Eight patients were identified by serological testing and showed more severe clinical manifestations. Two Ixodes persulcatus ticks detached from patients were positive for JMTV. All JMTV strains identified in this study formed a well-supported sub-lineage, distinct from those previously reported in China. Interpretation The public significance of JMTV should be highly concerning due to its potential pathogenicity for humans and efficient transmission by potential ticks. FUND: China Natural Science Foundation, State Key Research Development Programme, and United Kingdom Biotechnology and Biological Sciences Research Council.

The prevalence and clinical characteristics of tick-borne diseases at One Sentinel Hospital in Northeastern China.

Northeastern China is a region of high tick abundance, multiple tick-borne pathogens and likely human infections. The spectrum of diseases caused by tick-borne pathogens has not been objectively evaluated in this region for clinical management and for comparison with other regions globally where tick-transmitted diseases are common. Based on clinical symptoms, PCR, indirect immunofluorescent assay and (or) blood smear, we identified and described tick-borne diseases from patients with recent tick bite seen at Mudanjiang Forestry Central Hospital. From May 2010 to September 2011, 42% (75/180) of patients were diagnosed with a specific tick-borne disease, including Lyme borreliosis, tick-borne encephalitis, human granulocytic anaplasmosis, human babesiosis and spotted fever group rickettsiosis. When we compared clinical and laboratory features to identify factors that might discriminate tick-transmitted infections from those lacking that evidence, we revealed that erythema migrans and neurological manifestations were statistically significantly differently presented between those with and without documented aetiologies (P < 0.001, P = 0.003). Twelve patients (6.7%, 12/180) were co-infected with two tick-borne pathogens. We demonstrated the poor ability of clinicians to identify the specific tick-borne disease. In addition, it is necessary to develop specific laboratory assays for optimal diagnosis of tick-borne diseases.

Human Babesiosis Caused by a Babesia crassa-Like Pathogen: A Case Series.

Background: Human babesiosis is an emerging health problem in China. Methods: Babesia were identified in ticks, sheep, and humans in northeastern China using polymerase chain reaction (PCR) followed by genetic sequencing. We enrolled residents who experienced a viral-like illness after recent tick bite or were healthy residents. We defined a case using the definition for babesiosis developed by the US Centers for Disease Control and Prevention. Results: A Babesia crassa-like agent was identified in Ixodes persulcatus and Haemaphysalis concinna ticks using PCR followed by sequencing. The agent was characterized through phylogenetic analyses of the 18S rRNA gene, the ß-tubulin gene, and the internal transcribed spacer region. We tested sheep as a possible reservoir and found that 1.1% were infected with the B. crassa-like agent. We screened 1125 human participants following tick bites using B. crassa-specific PCR and identified 31 confirmed and 27 suspected cases. All the patients were previously healthy except for 1 with an ovarian tumor. Headache (74%), nausea or vomiting (52%), and fever (48%) were the most common clinical manifestations of confirmed cases. Six of 10 cases remained PCR positive for B. crassa-like infection 9 months after initial diagnosis. Asymptomatic infections were detected in 7.5% of 160 local residents. Conclusions: We identified B. crassa-like infection in people in northeastern China that caused mild to moderate symptoms. The possibility of more severe disease in immunocompromised patients and of transmission through the blood supply due to asymptomatic infections justifies further investigation of this reported infection.

Borrelia miyamotoi Infections in Humans and Ticks, Northeastern China.

We conducted an investigation of Borrelia miyamotoi infections in humans and ticks in northeastern China. Of 984 patients reporting recent tick bites, 14 (1.4%) were found to be infected with B. miyamotoi by PCR and genomic sequencing. The 14 patients had nonspecific febrile manifestations, including fever, headache, anorexia, asthenia, and arthralgia. Rash, eschar, and regional lymphadenopathy were each observed in 1 patient. Four (28.6%) patients were hospitalized because of severe disease. B. miyamotoi was detected in 3.0% (19/627) of Ixodes persulcatus, 1 (2.8%) of 36 Haemaphysalis concinna, and none of 29 Dermacentor silvarum ticks. Phylogenetic analyses based on sequences of a nearly entire 16s rRNA gene, a partial flagellin gene, and the glycerophosphodiester phosphodiesterase gene revealed that B. miyamotoi identified in patients and ticks were clustered in the group of the Siberian type. These findings indicate that B. miyamotoi is endemic in northeastern China and its public health significance deserves further investigation.

Human infection with a novel tick-borne Anaplasma species in China: a surveillance study.

BACKGROUND: Anaplasma phagocytophilum and Anaplasma ovis cause human infections. We investigated the potential for human pathogenicity of a newly discovered Anaplasma species infecting goats in China. METHODS: We collected blood samples from patients with a history of tick bite in the preceding 2 months at Mudanjiang Forestry Central Hospital of Heilongjiang Province from May 1, to June 10, 2014, to detect the novel Anaplasma species by PCR. We inoculated positive samples into cell cultures. We characterised the isolated pathogen by morphological and phylogenetic analyses. We tested serum antibodies by indirect immunofluorescence assay. FINDINGS: 28 (6%) of 477 patients assessed were infected with the novel Anaplasma species according to PCR and sequencing. We isolated the pathogen in vitro from three patients. Phylogenetic analyses of rrs, gltA, groEL, msp2, and msp4 showed that the pathogen was distinct from all known Anaplasma species. We provisionally nominate it "Anaplasma capra". 22 (92%) of 24 patients with data available had seroconversion or a four-fold increase in antibody titres. All 28 patients developed non-specific febrile manifestations, including fever in 23 (82%), headache in 14 (50%), malaise in 13 (46%), dizziness in nine (32%), myalgia in four (14%), and chills in four (14%). Additionally, ten (36%) of 28 patients had rash or eschar, eight (29%) had lymphadenopathy, eight (29%) had gastrointestinal symptoms, and three (11%) had stiff neck. Five patients were admitted to hospital because of severe disease. Six (35%) of 17 patients with data available had high hepatic aminotransferase concentrations. INTERPRETATION: The emergence of "A capra" as a cause of human disease suggests that individuals living in or travelling to endemic regions in northern China should take precautions to reduce their risk of exposure to this novel tick-borne pathogen. FUNDING: Natural Science Foundation of China and the US National Institutes of Health.

Epidemiological, clinical, and laboratory characteristics of 48 cases of "Babesia venatorum" infection in China: a descriptive study.

BACKGROUND: Human babesiosis is an emerging zoonosis. "Babesia venatorum" has been identified in only four asplenic men and a child so far. We aimed to describe the epidemiological, clinical, and laboratory characteristics of a series of cases with "B venatorum" infection identified in a sentinel hospital in China. METHODS: We recruited participants with a recent tick bite at Mudanjiang Forestry Central Hospital, Heilongjiang province, China. Cases were diagnosed through PCR followed by sequencing, microscopic identification, or isolation by animal inoculation, or both. FINDINGS: 48 individuals (30 women or girls; median age 45 years, range 7 months to 75 years) with "B venatorum" infection were identified. 32 of these individuals were confirmed cases and 16 were probable cases. None of the 48 cases had received a blood transfusion or had a splenectomy. Geographically, cases were distributed diffusely throughout the hospital catchment area. Of the 32 confirmed cases, 21 (66%) presented with a fever, 13 (41%) with a headache, 12 (38%) with myalgia or arthralgia, and three (9%) with chills. 14 (44%) patients had fatigue, eight (25%) had dizziness, and eight (25%) had hypersomnia. Six (19%) patients had an erythematous non-pruritic rash around the tick-bite site and two (6%) had lymphadenopathy. Seven (22%) and four (13%) patients had anaemia and thrombocytopenia, respectively, and seven (50%) of 14 patients with confirmed infection had increased hepatic transaminase concentrations. In the confirmed cases, concentrations of intercellular adhesion molecule 3 (p<0·001), P-selectin (p<0·05), and platelet endothelial cell adhesion molecule 1 (p<0·001) were significantly reduced, whereas tumour necrosis factor α (p<0·01) and vascular cell adhesion molecule 1 (p<0·001) were significantly increased. INTERPRETATION: "B venatorum" infection should be included in the differential diagnosis of patients with a tick-exposure history in areas where this pathogen has previously been identified in ticks or people. FUNDING: Natural Science Foundation of China and Mega-Project for Infectious Diseases.

Human infections with Rickettsia raoultii, China.

We used molecular methods to identify Rickettsia raoultii infections in 2 persons in China. These persons had localized rashes around sites of tick bites. R. raoultii DNA was detected in 4% of Dermacentor silvarum ticks collected in the same area of China and in 1 feeding tick detached from 1 patient.

Genome sequence of Borrelia afzelii Strain HLJ01, isolated from a patient in China.

We report here the genome sequence of Borrelia afzelii strain HLJ01, isolated from a patient with Lyme disease in China. It is the first report of the whole genome of a B. burgdorferi sensu lato isolate from a human in China.

Human infection with Candidatus Neoehrlichia mikurensis, China.

To identify Candidatus Neoehrlichia mikurensis infection in northeastern China, we tested blood samples from 622 febrile patients. We identified in 7 infected patients and natural foci for this bacterium. Field surveys showed that 1.6% of ticks and 3.8% of rodents collected from residences of patients were also infected.

[Dynamic investigation on the co-infection status of two pathogens in ticks from tourist point in Heilongjiang province].

OBJECTIVE: To monitor the co-infection status of Borrelia burgdorferi sensu lato (B.b.s.l) and spotted fever group Rickettsia (SFGR) in tourist areas of Heilongjiang province. METHODS: Polymerase chain reaction (PCR) was used to detect the 5S-23S rRNA intergenic spacer of B.b.s.l and ompA of SFGR in ticks, dynamically collected from tourist areas of Heilongjiang province in 2010. Amplification products from positive ticks were sequenced, and phylogenetic analysis was conducted by Mega 5.0 software package. RESULTS: 849 ticks were collected from two tourist points, with the dominant ticks in Tiger Mountain and Jingpo Lake were Ixodes persulcatus and Haemaphysalis concinna. Regarding the Ixodes persulcatus from Tiger Mountain, the infection rates of B.b.s.l and SFGR were 26.15% and 10.05%. The infection rate of SFGR was 13.33% in Haemaphysalis concinna and the B.b.s.l was undiscovered in the same ticks from Jingpo Lake. However, the co-infection could only be detected in Ixodes persulcatus of both tourist areas. Surveillance data showed that the major ticks were more likely to be appeared in July at Tiger Mountain and in June at Jingpo Lake. Data from the sequence analysis on B.b.s.l showed that the B.b.s.l in tourist areas could be classified into three different genotypes, other than B. garinii and B. afzelii. We first detected B. valaisiana-like group genotype in northeast of China. Results from the sequence analysis of SFGR positive products showed that the two DNA sequences of newly detected agents were completely the same as Rickettsia sp. HL-93 which was detected in Hulin and Rickettsia sp. H820 found in northeast, China. CONCLUSION: The co-infection of B.b.s.l and SFGR was detected in ticks from the tourist areas of Heilongjiang province, and data from the sequencing of specific fragment showed that various kinds of genotypes existed in this area. However; the rates of co-infections-different according to environment, time and population that contributed to the kinds of and the index of ticks existed in the surveys points, also the infection rate of the ticks was studied.

Complete genomic characterization of two tick-borne encephalitis viruses isolated from China.

Tick-borne encephalitis (TBEV) is prevalent over a wide area of the Eurasian continent. TBE viruses cause severe encephalitis in humans, with serious sequelae, and have a significant impact on public health in these endemic regions. To gain insight into genetic evolution of tick-borne encephalitis virus (TBEV) in China, the complete genomic sequences of two TBEV strains Senzhang and MDJ01, which were isolated in 1953 and 2001 respectively, were characterized. The complete genome sequences of two strains were all consist 10,784 nucleotides and there are 364 nucleotides deletion in the 3' nontranslated region. Compared with other TBEV strains, homology range from 85.2% (Zausaev) to 99.6% (MDJ02 and MDJ03) on the level of nucleotide. Phylogenetic trees based on the complete genome, open reading frame and E gene nucleotide sequences all showed that the strains Senzhang and MDJ01 belong to Far-Eastern subtype and cluster with other Chinese TBEV strains. All these implied that TBEVs prevalent in China were highly conservative, other measurement should be taken to improve protective efficacy of present vaccine.

Complete genome sequence analysis of tick-borne encephalitis viruses isolated in northeastern China.

Tick-borne encephalitis virus (TBEV) causes lethal encephalitis in humans, posing a growing public-health problem in many European and Asian countries. TBEV is currently endemic in northeastern China, but the complete genome sequences of Chinese TBEV strains have not been reported. During a TBE outbreak in 2010 in Mudanjiang City, Heilongjiang Province, China, two TBEV strains were isolated from serum samples of two patients, and the complete sequences were determined and compared with other known TBEV strains. Both Mudanjiang isolates consisted of 10,774 nucleotides and encoded a single open reading frame coding for a polyprotein of 3414 amino acids, and a unique deletion of 364 nucleotides in the 3' untranslated region (UTR) was recorded. Phylogenetic analysis based on the amino acid sequence of the E protein and the nucleotide sequence of the 3'UTR revealed that the Mudanjiang isolates are closely related to the Senzhang and Sofjin-HO strains and belong to the Far Eastern subtype of TBEV. These findings provide insight into the evolutionary relationships among Chinese TBEV isolates and are useful for laboratory diagnosis and vaccine development for TBEV.