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PCR amplification of the rrs2 gene indicated that 50% (62/124) of insectivorous bats from eastern China were infected with Leptospira borgpetersenii, L. kirschneri, and several potentially new Leptospira species. Multilocus sequence typing defined 3 novel sequence types in L. kirschneri, suggesting that bats are major carriers of Leptospira.

We isolated Rickettsia japonica from a febrile patient in Lu’an City, China, in 2013. Subsequently, we found an R. japonica seroprevalence of 54.8% (494/902) in the rural population of Anhui Province and an R. japonica prevalence in Haemaphysalis longicornis ticks of 0.5% (5/935). R. japonica and its tick vector exist in China.

PCR amplification indicated the minimum infection rate of Rickettsia spp. was 0.66% in Haemaphysalis longicornis ticks collected from Shandong Province, China. Phylogenetic analysis based on the rrs, gltA, ompA, and ompB genes indicated that the ticks carried R. japonica, Candidatus Rickettsia longicornii, and a novel Rickettsia species related to R. canadensis.

Severe fever with thrombocytopenia syndrome is an emerging hemorrhagic fever disease in eastern Asia, caused by a tickborne bunyavirus. Of 25 patients hospitalized with this disease in China, 100% produced and maintained neutralizing antibodies to severe fever with thrombocytopenia syndrome virus for the study period of 4 years.

Supportive therapy is recommended before laboratory confirmation of this disease., Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease in China. The incidence and clinical and laboratory characteristics of SFTS are not clearly defined. During May 22–October 2, 2011, a total of 24 patients with fever, thrombocytopenia, and leukopenia were clinically diagnosed as having SFTS in Yiyuan County, Shandong Province, China. We conducted laboratory tests for these SFTS patients. SFTS virus (SFTSV) infection was confirmed in 22 patients by using reverse transcription PCR and ELISA by acute-phase and convalescent-phase serum samples. Clinical and laboratory manifestations included fever (100%), gastrointestinal symptoms (91%), myalgia (55%), chills (41%), thrombocytopenia (100%), and leukopenia (95%).

Analysis of hemorrhagic fever with renal syndrome cases in Zibo City, China, during 2006-2014 showed that it occurred year-round. Peaks in spring and fall/winter were caused by Hantaan and Seoul viruses, respectively. Rodent hosts were the striped field mouse for Hantaan virus and the brown rat and house mouse for Seoul virus.

To evaluate the role of small mammals as hosts of severe fever with thrombocytopenia syndrome virus (SFTSV), we tested serum samples from rodents and shrews in China, collected in 2013. SFTSV antibodies and RNA were detected, suggesting that rodents and shrews might be hosts for SFTSV.

We developed a typing method that can differentiate 8 strains of Rickettsia prowazekii into 7 genotypes. This method can be used to type and trace the origin of R. prowazekii isolated from samples collected during epidemics after a bioterrorism attack.

To the Editor: Hemorrhagic fever with renal syndrome (HFRS) is caused by hantavirus. During 1995–2005, China reported 20,000–50,000 cases of HFRS annually, which represents 90% of HFRS cases worldwide (1–3). In China, HFRS is caused mainly by 2 serotypes of hantavirus: Hantaan virus (HTNV) and Seoul virus (SEOV) (4). Pathogenic hantavirus serotypes are considered to be strictly associated with their serotype-specific reservoir hosts. HTNV is associated with the striped field mouse (Apodemus agrarius), and SEOV is associated with the brown rat (Rattus norvegicus) and the black rat (Rattus rattus) (4,5). HTNV causes a severe form of HFRS, characterized by renal failure that in some cases is followed by pulmonary edema and disseminated intravascular coagulation; the estimated death rate is 5%–15%. SEOV causes a moderate form of HFRS (6). Jiaonan County in Shandong Province is one of the high-incidence HFRS areas in China. To detect the hantavirus infection in small mammals, we trapped rodents and shrews during December 2012–November 2013 using snap-traps in Jiaonan County (longitude 119°30′–120°30′, latitude 35°35′–36°08′). We captured 1,276 animals comprising 5 rodent species and 1 shrew species (Table) and analyzed serum antibody against hantavirus of each animal using an antigen sandwich ELISA Kit (Shanghai Jiahe Biotechnology, Shanghai, China). The serum was considered to contain antibodies against hantavirus when the optical density (OD)450nm of the sample was greater than the threshold. The threshold was calculated by using the equation: threshold = the average OD of the negative control + 0.15. ELISA results showed that 23.3% of animals were seropositive to hantavirus antigen (Table). The seropositive rate to hantavirus was 44.0% in Asian house shrews (Suncus murinus), 25.3% in house mice (Mus musculus), 15.4% in Chinese hamsters (Cricetulus griseus), 10.3% in brown rats, 10.1% in striped field mice (Apodemus agraius), and 3.0% in greater long-tailed hamsters (C. triton). The seropositivity rate for rodents was higher during summer (May–August) and lower during spring (March and April) and winter (October and November) but not significantly different among the months. Table Seropositive rate and RT-PCR–positive rate of hantaviruses in small mammals, Jiaonan County, China, December 2012–November 2013 To determine what types of hantavirus infected the animals, we amplified viral RNA of HTNV and SEOV from animal lung samples using reverse transcription PCR with serotype-specific primers (7); 2.1% of animals had viral RNA of HTNV, and 2.1% had viral RNA of SEOV (Table). HTNV RNA was detected in striped field mice (6.3%), house mice (1.4%), and brown rats (0.6%). The hantavirus-positive animals were captured in February, April, and November for stripped field mice; November for brown rats; and April and November for house mice. SEOV was detected in brown rats (8.2%) and Asian house shrews (1.7%). These SEOV-positive animals were captured in January, March, May, June, and July for brown rats and March and November for Asian house shrews. The phylogenetic analysis of sequences amplified by reverse transcription PCR is presented in the Technical Appendix Figure. The nucleotide sequences of the PCR products have been deposited in GenBank (accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"KM357423-KM357452","start_term":"KM357423","end_term":"KM357452","start_term_id":"725611688","end_term_id":"723941204"}}KM357423-KM357452). Hantavirus had been considered to be strictly associated with specific reservoir hosts and to have the same geographic distribution pattern as these reservoir hosts. All hantaviruses that caused human diseases had been associated with rodents, including members of Murinae, Arvicolinae, and Sigmodontinae spp. Insectivore hantaviruses were not known to cause human disease. The rodent hantavirus and the insectivorous hantaviruses were thought to have co-evolved with their specific rodent and insectivorous hosts over millions of years (8). One observed geographic clustering of hantavirus strains, and the association of hantaviruses with their reservoirs, might have been caused by an isolation-by-distance mechanism (9,10) and mixture of both host switching and co-divergence (10). Our study demonstrated that HTNV not only infects its traditional host, the striped mouse, but also infects house mice and rats; SEOV infects not only rats but also shrews, suggesting host expansion for both HTNV and SEOV in China. Our hypothesis is that the hantaviruses co-evolved with their animal hosts, such as SEOV with rats and HTNV with striped mice, but when their animal hosts expanded their territory, hantavirus had more chance to infect other susceptible rodents and expanded their animal hosts. Both Asian house shrews and house mice are closely associated with humans by living inside and outside of human houses in China. The Asian house shrew and house mouse have been underestimated as potential animal hosts of SEOV and HTNV. To our knowledge, only 1 previous study had associated Asian house shrews with SEOV; in that study, an SEOV strain was isolated from an Asian house shrew in China (2). Technical Appendix: Phylogenetic tree of hantaviruses from small mammals, Jiaonan County, China, December 2012–November 2013. Click here to view.(98K, pdf)

Well-documented cases of simultaneous human infection with more than one tick-borne pathogen are rare. To our knowledge only two dual infections have been reported: simultaneous human infection with the agent of human granulocytic ehrlichiosis and Borrelia burgdorferi and simultaneous human infection with B. burgdorferi and Babesia microti (1-2). Rocky Mountain spotted fever has long been known to be endemic in North Carolina; cases of human ehrlichial infection were recognized there soon after Ehrlichia chaffeensis was recognized as an important cause of tick-borne disease in the southeastern United States. Because both Rocky Mountain spotted fever and ehrlichiosis are prevalent in North Carolina, occasional cases of simultaneous human infection by rickettsial and ehrlichial agents would not be surprising; however, no such cases seem to have been reported.

Severe fever with thrombocytopenia syndrome, which results in severe illness and has a high case-fatality rate, is caused by a novel bunyavirus, severe fever with thrombocytopenia syndrome virus. We found that samples from 2/237 (0.8%) healthy persons and 111/134 (83%) goats in Yiyuan County, Shandong Province, China, were seropositive for this virus.

Suggested citation for this article: Long SW, Pound JM, Yu X. Ehrlichia prevalence in Amblyomma Ehrlichia chaffeensis and E. ewingii, agents of human monocytic ehrlichiosis and ehrlichiosis ewingii, respectively, are transmitted by the lone star tick, Amblyomma americanum, which is found from west-central Texas northward to Iowa, and southeastward to the Atlantic Coast (1). In A. americanum, E. chaffeensis has been found in several states, while E. ewingii has only been found in North Carolina, Florida, and Missouri (1,2). E. ewingii infection in white-tailed deer (Odocoileus virginianus), a potential reservoir, has been found in the states mentioned previously as well as in Kentucky, Georgia, and South Carolina (3,4). Human ehrlichioses are underdiagnosed in the United States and may be as prevalent as Rocky Mountain spotted fever in some areas (1). Ehrlichioses are prevalent in Texas, and fatal cases have been reported (1,5). This study was conducted to examine ticks from central Texas for Ehrlichia and provide information to increase public health awareness of this problem. Adult A. americanum ticks were collected from a 38.8-hectare game fenced-pasture (Plot #8) in the Kerr Wildlife Management Area, Kerr County, Texas. Ticks were trapped by using blocks (approximately 85 g) of dry ice centered on smooth, white, nylon cloths measuring approximately 1 m2. These traps were placed on the ground in the brush or in areas under tree canopies for approximately 1 h. Trapped adult A. americanum were frozen in liquid nitrogen and then bisected with a sterile scalpel. Halves of the bisected ticks were stored at –80oC. The other halves were pooled in groups of six. DNA was extracted from these pools by using the QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA), and evaluated by using a nested species-specific 16S rRNA gene polymerase chain reaction (PCR) for E. chaffeensis and E. ewingii, with E. canis as a negative control. The first-round primers were genus-specific for Ehrlichia (ECC and ECB). The forward primers of the nested PCR were HE1, EE72, and Ecan, which were specific to E. chaffeensis, E. ewingii, and E. canis, respectively. The reverse primer is a common primer (HE3) for all species (6–8). An aliquot of the negative control reaction containing no DNA template was carried through both rounds of the nested PCR with every reaction set. A dilution series of stock E. chaffeensis DNA mixed with tick DNA showed no substantial inhibition of the PCR, even with DNA concentrations as low as 0.2 ng/mL. Tick pools positive for E. chaffeensis or E. ewingii by PCR were examined by using DNA from the individual tick halves. DNA was extracted by using the Nucleobond DNA/RNA Isolation Kit (BD Biosciences Clontech, Palo Alto, CA). To confirm positive PCR results for individual ticks, first-round amplicons (primers ECB and ECC) were separated by electrophoresis. The 478-bp band was recovered using the QIAquick Gel Extraction Kit, then cloned into the pCR2.1-TOPO vector with the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). DNA sequences were obtained from both directions of the insert in the recombinant plasmids by using PE Applied Biosystems (Foster City, CA) 373XL automated DNA sequencers in the UTMB Sequencing Core. Of the 66 adult A. americanum ticks examined, 5 were positive for E. ewingii (7.6%). The 16S rRNA gene sequences from these five positive samples were most similar to the E. ewingii 16S rRNA gene sequence (GenBank accession no.{"type":"entrez-nucleotide","attrs":{"text":"U96436","term_id":"2352090","term_text":"U96436"}}U96436). Sequence variations are summarized in the Table. These mutations may result from polymerase errors prior to cloning. E. ewingii has never been cultured or handled by our laboratory, and all negative controls for the nested PCR were negative, minimizing the possibility of false-positive results. Table Sequence variation of the 16S rRNA gene in Ehrlichia ewingii detected in Amblyomma americanum ticks from central Texas, compared to the partial 16S rRNA gene sequence of Ehrlichia ewingii in Genbank (accession {"type":"entrez-nucleotide","attrs":{"text":"U96436","term_id":"2352090","term_text":"U96436"}} ... This is the first report of ticks infected with E. ewingii in states other than North Carolina, Florida, or Missouri. Ticks are found in damp wooded areas (1,9). Seasonal population changes have been associated with climatic factors, including precipitation, temperature, and day length (9–11). These ticks were collected during August, one of the hottest months of the year in Texas, with temperatures averaging 33°C. Adult ticks are more abundant earlier in the summer, and the actual prevalence of E. ewingii infection may be higher. August is a dry month in Texas, averaging 2.32 inches of an annual rainfall of 26 to 30 inches (12). Although cases of E. ewingii infection have not been reported from Texas, this study shows the presence of ticks infected with E. ewingii in Texas. No ticks infected with E. chaffeensis were found in this sample. The prevalence of E. chaffeensis may be so low that it was not detected in the small sample size. Also, E. chaffeensis may not survive well at this extreme of the host range. Infection exclusion may occur in the tick or reservoir hosts (or both), such that an established population of one ehrlichial species prevents another ehrlichial species from establishing itself. This phenomenon has been noted in the related rickettsial organisms Rickettsia peacockii and R. rickettsii in the Rocky Mountain wood tick, Dermacentor andersoni (13). Another finding involves using nested 16S rRNA PCR to identify ehrlichial infection. These primers are not as specific as thought previously. Arthropods should be carefully cleaned to prevent contamination by Shigella and other soil contaminants. A single positive-nested PCR reaction should not be considered sufficient for positive identification of the organism. Sequencing of the outer PCR product, or another confirming method, should be used to positively identify the organism. Primers directed to more divergent sequences, such as the dsb gene, should be utilized in place of, or in addition to, 16S rRNA gene PCR (14). This study was supported by a fellowship for Scott W. Long from the Sealy Center for Vaccine Development, University of Texas Medical Branch, and a grant from the National Institute of Allergy and Infectious Diseases (AI45871).