During May 2–July 30, 2010, a total of 622 febrile patients, who had histories of recent tick bites and sought treatment at Mudanjiang Forestry Central Hospital () were screened for the infections of tick-borne agents. When patients were admitted, peripheral blood samples were collected and treated with EDTA. DNA as extracted by using the QIAmp DNA Blood Mini Kit (QIAGEN, Germantown, MD, USA).
Location of Mudanjiang, Heilongjiang Province, China, where Candidatus Neoehrlichia mikurensis was detected.
For a broad-range assay, a nested PCR specific for the 16S rRNA gene (rrs
) was used to detect organisms in the family Anaplasmataceae
. For positive samples, 2 heminested PCRs were used to amplify the entire rrs
gene. For further confirmation, a nested PCR specific for the 60-kDa heat shock protein gene (groEL
) was performed. Detailed cycling conditions for all amplifications are described in the Technical Appendix
Seven patients were found to be infected with Candidatus
N. mikurensis by amplifications of the rrs
genes. Amplified rrs
gene (1,501 bp) and partial groEL
gene (1,230 bp) sequences from these patients were identical. These sequences were also identical to genes of Candidatus
N. mikurensis detected in ticks and rodents in the Asian region of Russia (5
Serum samples were collected from patients during the acute (2–12 days after onset of illness) or convalescent (34–42 days after onset of illness) phases of illness. All samples were negative for IgG against Anaplasma phagocytophilum, Ehrlichia chaffeensis, Borrelia burgdorferi, Rickettsia heilongjiangensis, and tick-borne encephalitis virus when tested by indirect immunofluorescence assay.
All 7 patients were farmers residing in the villages in Mudanjiang. Their median age was 41 years (range 29–67 years) and 5 were men. None had been vaccinated against tick-borne encephalitis. The patients had onset of illness during May 20–July 13, 2010. The median time from the tick bite to the onset of illness and from the onset of illness to the physician visit was 8 days (range 2–35 days) and 7 days (range 1–12 days), respectively.
All patients were otherwise healthy, and none had a history of underlying immunocompromised conditions. Fever, headache, and malaise were reported for all 7 patients. Other major manifestations included nausea (5/7), vomiting (5/7), myalgia (4/7), and stiff neck (4/7). Less common symptoms were arthralgias (2/7), cough (2/7), diarrhea (1/7), confusion (1/7), and erythema (1/7). Skin erythema (multiple and oval) was seen on the neck of 1 patient.
Laboratory test results showed leukopenia in 1 patient, leukocytosis in 1 patient, thrombocytopenia in 2 patients, and anemia in 2 patients. Serum levels of alanine aminotransferase and aspartate aminotransferase were within reference ranges for all patients. Wright–Giemsa stained peripheral blood smears did not show morulae or other blood parasites.
To identify local natural foci, we performed a field investigation on infections of Candidatus N. mikurensis in ticks and rodents from areas of residences of the patients. During May–July 2010, a total of 516 host-seeking ticks, including 316 I. persulcatus, 187 Haemaphysalis concinna, and 13 Dermacentor silvarum, were collected on vegetation and individually examined. Candidatus N. mikurensis DNA was detected in 6 (1.9%) I. persulcatus and 2 (0.8%) H. concinna ticks, but no DNA was detected in D. silvarum ticks ().
Prevalence of Candidatus Neoehrlichia mikurensis in ticks and rodents, Mudanjiang, China
A total of 211 rodents of various species were captured by using snap traps. After rodent species was identified, spleen specimens were collected for DNA extraction and PCR. Eight rodents of 3 species, 5 (4.6%) Clethrionomys rufocanus, 2 (5.7%) Rattus norvegicus, and 1 (33.3%) Tamias sibiricus, were positive for Candidatus N. mikurensis ().
Nucleotide sequences of rrs and groEL genes of 8 ticks and 8 rodents were identical to each other and to sequences obtained from the 7 patients. Phylogenetic analysis of rrs genes showed that nucleotide sequences identified were identical to those of Candidatus N. mikurensis from Japan and the Asian region of Russia but different from sequences from Europe (99.6%–99.8% similarity) (, panel A). Similar phylogenetic relationships were observed in a neighbor-joining tree based on groEL gene nucleotide sequences. In comparison with sequences from humans and ticks in Europe, the groEL gene sequences identified in the study showed 97.6%–98.4% similarity (, panel B).
Figure 2 A) Neighbor-joining trees based on the 16S rRNA gene (rrs) and B) the 60-kDa heat shock protein gene (groEL) of Candidatus Neoehrlichia mikurensis, China, generated by using Molecular Evolutionary Genetics Analysis software version 4.0, (www.megasoftware.net/ (more ...)