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.

First Detection of Mukawa Virus in Ixodes persulcatus and Haemaphysalis concinna in China.

Mukawa virus (MKWV), a novel tick-borne virus (TBV) of the genus Phlebovirus of family Phenuiviridae, has been firstly reported in Ixodes persulcatus in Japan. In this study, we made an epidemiological investigation in China to obtain the geographic distribution and genetic features of this virus outside Japan. We screened 1,815 adult ticks (665 I. persulcatus, 336 Dermacentor silvarum, 599 Haemaphysalis longicornis, 170 Rhipicephalus microplus, 45 Haemaphysalis concinna) and 805 wild small mammals collected from eight provinces. The positive rate of 6.77% (45/665, including 18 female and 27 male I. persulcatus) and 2.22% (1/45, 1 male H. concinna) were obtained from I. persulcatus and H. concinna in Heilongjiang province, respectively. No evidence of MKWV infection was found in other three tick species or any of the mammalian species. The virus can infect the Vero cells successfully, indicating the ability of MKWV to replicate in mammalian cells. A phylogenetic tree based on the nucleotide sequences of L, M, and S segments demonstrated that the Japanese MKWV variant, our two MKWV variants, and KURV were clustered with the members of the mosquito/sandfly-borne phleboviruses and distant from other tick-borne phenuiviruses. A phylogenetic analysis based on 895 bp partial L gene sequences (n = 46) showed that all MKWV sequences were separated into three lineages. Our results showed the presence of MKWV in I. persulcatus and H. concinna in northeast of China, highlighting the necessity of epidemiological study in wider regions. Due to the ability of MKWV to replicate in mammalian cells, the potential for zoonosis, and wide distribution of I. persulcatus and H. concinna in China, the important vectors of MKWV, further screening to more tick species, wild animals, domestic animals, and humans raises up practical significance.

Identification of Myoferlin, a Potential Serodiagnostic Antigen of Clonorchiasis, via Immunoproteomic Analysis of Sera From Different Infection Periods and Excretory-Secretory Products of Clonorchis sinensis.

Clonorchiasis, which is caused by Clonorchis sinensis, is an important foodborne disease worldwide. The excretory-secretory products (ESPs) of C. sinensis play important roles in host-parasite interactions by acting as causative agents. In the present study, the ESPs and sera positive for C. sinensis were collected to identify proteins specific to the sera of C. sinensis (i.e., proteins that do not cross-react with Fasciola hepatica and Schistosoma japonicum) at different infection periods. Briefly, white Japanese rabbits were artificially infected with C. sinensis, and their sera were collected at 7 days post-infection (dpi), 14 dpi, 35 dpi, and 77 dpi. To identify the specific proteins in C. sinensis, a co-immunoprecipitation (Co-IP) assay was conducted using shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) to pull down the sera roots of C. sinensis, F. hepatica, and S. japonicum. For the annotated proteins, 32, 18, 39, and 35 proteins specific to C. sinensis were pulled down by the infected sera at 7, 14, 35, and 77 dpi, respectively. Three proteins, Dynein light chain-1, Dynein light chain-2 and Myoferlin were detected in all infection periods. Of these proteins, myoferlin is known to be overexpressed in several human cancers and could be a promising biomarker and therapeutic target for cancer cases. Accordingly, this protein was selected for further studies. To achieve a better expression, myoferlin was truncated into two parts, Myof1 and Myof2 (1,500 bp and 810 bp), based on the antigenic epitopes provided by bioinformatics. The estimated molecular weight of the recombinant proteins was 57.3 ku (Myof1) and 31.3 ku (Myof2). Further, both Myof1 and Myof2 could be probed by the sera from rabbits infected with C. sinensis. No cross-reaction occurred with the positive sera of S. japonica, F. hepatica, and negative controls. Such findings indicate that myoferlin may be an important diagnostic antigen present in the ESPs. Overall, the present study provides new insights into proteomic changes between ESPs and hosts in different infection periods by LC-MS/MS. Moreover, myoferlin, as a biomarker, may be used to develop an objective method for future diagnosis of clonorchiasis.

Genetic Diversity and Coexistence of Babesia in Ticks (Acari: Ixodidae) from Northeastern China.

Background: Human babesiosis is an emerging zoonotic disease transmitted by ticks in China. A few systematic reports on Babesia spp. was involved with ticks, especially in the human babesiosis endemic areas in Northeastern China. Materials and Methods: Ticks were collected from 30 individual waypoints along 2.0 km transects in two recreational forests. Babesia spp. infection in ticks was screened by amplifying the partial 18s rRNA gene with subsequent sequencing. Multivariate logistic regression analysis was used to determine the association between tick infection and related environmental risk factors. Cluster analyses were performed using SaTScan v6.0 software to identify any geographical cluster of infected ticks. Results: A total of 2380 Ixodes persulcatus and 461 Haemaphysalis concinna ticks were collected. Of the 0.97% of I. persulcatus ticks that tested positive, five Babesia species were identified, including B. bigemina (n = 6), B. divergens (n = 2), B. microti (n = 3), B. venatorum (n = 11), and one novel strain HLJ-8. Thirteen (2.92%) H. concinna ticks tested positive for B. bigemina (n = 1), B. divergens (n = 1), three genetic variants of Babesia represented by HLJ-874, which was closely related to Babesia sp.MA#361-1, and eight other Babesia variants represented by HLJ242, which were similar to B. crassa. Each study site had 5-6 different Babesia spp. One waypoint was more likely to yield B. venatorum (relative risk = 15.36, p = 0.045) than all other waypoints. Conclusions: There exists a high genetic diversity of Babesia spp. across a relatively small sampled region. Further study is needed to understand the risks these variants pose to human health.

Anaplasma platys-Like Infection in Goats, Beijing, China.

As one of the important tick-borne zoonotic pathogens, Anaplasma has both veterinary and public health significance. Here, we performed a survey of Anaplasma infection in the goats from a farm in Beijing, China, and found 44.6% (41/92) were infected with Anaplasma capra, and 22.8% (21/92) were infected with Anaplasma sp. This Anaplasma sp. bacterium was close to a recently emerging Anaplasma platys strain based on gltA and groEL gene phylogenetic analysis. As to further understand the characteristics of Anaplasma sp., we raised a couple of positive goats (n = 2) in the laboratory with tick-free settings. We observed inappetence, vomiting, high fever, and weakness of limbs in the goat's offspring (n = 3). In addition, the blood samples from all offspring were all positive of this Anaplasma spp. We did not see any intracellular morulae in neutrophils, monocytes, and erythrocytes, but we identified some in the platelets of the blood smears from the positive goats by light microscopy. We named it A. platys-like and suggested it may infect platelets and be transmitted vertically through the placenta of goats. These findings deserve further evaluation.

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.

Genetic diversity of Anaplasma and Ehrlichia bacteria found in Dermacentor and Ixodes ticks in Mongolia.

Anaplasma and Ehrlichia are tick-borne bacterial pathogens that cause human granulocytic anaplasmosis, human monocytic ehrlichiosis, and are severe threats to livestock economies like Mongolia. In this study, ticks were collected, identified, and pooled (n = 299) from three distinct environments across central Mongolia. Each pool was initially tested for Anaplasma/Ehrlichia using a 16S rRNA PCR assay that detects both genera, and specific PCR testing was done to identify those positive samples. Maximum likelihood estimation (MLE) of infection rates of ticks collected from the environment in Selenge aimag (province) found infection rates of Ixodes persulcatus ticks to be 2.0% (95% CI: 0.7, 4.3%) for A. phagocytophilum and 0.8% (95% CI: 0.1, 2.5%) for both nonspecific Ehrlichia and Anaplasma. Ehrlichia muris was only detected in I. persulcatus ticks collected from the Selenge aimag, where the MLE was 1.2% (95% CI: 0.1, 2.5%). The calculated MLE infection rate of Anaplasma spp. in questing Dermacentor nuttalli ticks ranged from 1.9% (95% CI: 1.1, 9.1%) in the Tov aimag to 2.3% (95% CI: 1.3, 10.8%) in the Selenge aimag. However, when examining MLE in ticks removed from livestock, estimates increase substantially, ranging from 7.8% (95% CI: 4.2, 13.3%) in Dornogovi to 22.5% (95% CI: 14.3, 34.3%) in Selenge, suggesting that livestock play a key role in disease maintenance. Considering the collective economic losses that can result from these pathogens and the potential for illness in nomadic herdsmen, these results highlight the need for enhanced TBD surveillance and prevention measures within Mongolia.

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.

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.

Distribution and molecular characteristics of rickettsiae found in ticks across Central Mongolia.

BACKGROUND: Little is known regarding tick-borne diseases in Mongolia, despite having 26% of the population still living nomadic pastoral lifestyles. A total of 1497 adult unfed ticks: 261 Ixodes persulcatus, 795 Dermacentor nuttalli, and 441 Hyalomma asiaticum, were collected from three ecologically distinct regions in Central Mongolia. Tick pools (n = 299) containing ~5 ticks each, were tested for Rickettsia and Tick-borne encephalitis virus (TBEV) using nested polymerase chain reaction, reverse transcription-PCR, and quantitative real-time RT-PCR. RESULTS: Assays yielded pooled prevalence of 92.5% (49/53) and 1.9% (1/53) for pooled I. persulcatus testing positive for "Candidatus Rickettsia tarasevichiae" and TBEV, respectively, while Rickettsia raoultii was found in 72.8% (115/158) of pooled D. nuttalli samples. When calculating a maximum likelihood estimation, an estimated 46.6% (95% CI: 35.2-63.6%) of I. persulcatus ticks in the pooled sample were infected with "Candidatus R. tarasevichiae". CONCLUSIONS: Both "Candidatus R. tarasevichiae" and R. raoultii are recognized as emerging tick-borne pathogens, with this being one of the first reports of "Candidatus R. tarasevichiae" in Mongolia. Given that "Candidatus R. tarasevichiae" shares the same vector (I. persulcatus) as TBEV, and infections may present with similar symptoms, Mongolian physicians treating suspected cases of TBEV should include "Candidatus R. tarasevichiae" infection in their differential diagnosis and consider prescribing antimicrobial therapy.

Transmission dynamics of Ebola virus disease and intervention effectiveness in Sierra Leone.

Sierra Leone is the most severely affected country by an unprecedented outbreak of Ebola virus disease (EVD) in West Africa. Although successfully contained, the transmission dynamics of EVD and the impact of interventions in the country remain unclear. We established a database of confirmed and suspected EVD cases from May 2014 to September 2015 in Sierra Leone and mapped the spatiotemporal distribution of cases at the chiefdom level. A Poisson transmission model revealed that the transmissibility at the chiefdom level, estimated as the average number of secondary infections caused by a patient per week, was reduced by 43% [95% confidence interval (CI): 30%, 52%] after October 2014, when the strategic plan of the United Nations Mission for Emergency Ebola Response was initiated, and by 65% (95% CI: 57%, 71%) after the end of December 2014, when 100% case isolation and safe burials were essentially achieved, both compared with before October 2014. Population density, proximity to Ebola treatment centers, cropland coverage, and atmospheric temperature were associated with EVD transmission. The household secondary attack rate (SAR) was estimated to be 0.059 (95% CI: 0.050, 0.070) for the overall outbreak. The household SAR was reduced by 82%, from 0.093 to 0.017, after the nationwide campaign to achieve 100% case isolation and safe burials had been conducted. This study provides a complete overview of the transmission dynamics of the 2014-2015 EVD outbreak in Sierra Leone at both chiefdom and household levels. The interventions implemented in Sierra Leone seem effective in containing the epidemic, particularly in interrupting household transmission.

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.