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in 20022003 in kochi prefecture , western japan , 2 men sought medical care for rickettsiosis - like signs and symptoms .
case - patient 1 ( 61 years old ) sought care for fever ( 39.2c ) , chills , and malaise 10 days after traveling to the mountains ( day 0 , the day of symptom onset ) .
his physician prescribed cefdinir ( 300 mg / day ) . by day 3 , signs and symptoms had not improved and an erythematous rash on his trunk had spread ; the physician suspected infection with r. japonica or o. tsutsugamushi . the patient was hospitalized and intravenously administered minocycline ( 200 mg / day ) .
results ( and reference values ) for laboratory tests ( day 3 ) follow : leukocytes , 5.8  10 cells / l ( 3.59.2  10 cells / l ) ; thrombocytes , 225 
l ) ; aspartate aminotransferase , 59 u / l ( < 38 u / l ) ; alanine aminotransferase , 61 u / l ( < 36 u / l ) ; and c - reactive protein , 12.1 mg / dl ( < 0.3 mg / dl ) .
case - patient 2 , a 73-year - old lumberjack , sought medical care for fever ( 39.2c ) , headache , and malaise ( day 0 , the day of symptom onset ) . on day 4 ,
a disseminated maculopapular rash was noticed , especially on the trunk and lower limbs ; jsf or scrub typhus infection was suspected .
10 cells / l ; aspartate aminotransferase , 100 u / l , alanine aminotransferase , 45 u / l ; and c - reactive protein , 17.2 mg / dl . in 2003 , blood clots and serum samples from the 2 patients were transferred from kochi institute of health to the university of shizuoka , where they were stored at 20c until a retrospective analysis could be performed .
dna was extracted from the blood clots , and nested pcr was performed , as described ( 3,9 ) , to detect sfg rickettsiae 16s rdna , o. tsutsugamushi 16s rdna , a. phagocytophilum p44/msp2 , and ehrlichia spp .
p28/omp-1 ( table 1 ) . to avoid dna contamination , we performed pcr , electrophoresis , and cloning were performed in separate laboratories . as a negative control , nested pcr without dna template samples was performed for each sample .
pcr detected a. phagocytophilum p44/msp2 multigenes in acute - phase blood clots from both case - patients , and sfg rickettsia 16s rdna was amplified from a sample from case - patient 2 ( table 1 ) .
after in - hospital treatment with minocycline ( 200 mg / d ) , both case - patients improved clinically and were discharged on days 20 and 12 , respectively , after symptom onset . 
before being used in pcr , blood clots from the patients were homogenized by using biomasher ( nippi inc . ,
tokyo , japan ) and treated overnight with 100 u of streptokinase ( wako pure chemical industries ltd , osaka , japan ) .
dna then was extracted by using the qiaamp dna mini kit ( qiagen , valencia , ca , usa ) .
multiplex nested first - step pcr for sfg rickettsiae and o. tsutsugamushi was performed by using the following primers : ro-1f ( 5'-ccgtaaacgatgagtgctaga-3 ' ) and ro - r1 ( 5'-ccgagaacgtattcaccgc-3 ' ) .
multiplex nested second - step pcr for sfg rickettsiae 16s rdna was performed by using the following primers : r-2f ( 5'-gaagattctctttcggtttcgc-3 ' ) and r-2r ( 5'-gtcttgcttccctctgtaaac-3 ' ) .
multiplex nested second - step pcr for o. tsutsugamushi 16s rdna was performed by using the following primers : o-2f ( 5'-gacatggtagtcgcgaaaaatg-3 ' ) and o-2r ( 5'-tgcaatccgaactgagatacc-3 ' ) .
a. phagocytophilum p44/msp2 was amplified by using primers p3726 , p4257 , p3761 , and p4183 , and ehrlichia spp .
p28/omp-1 was amplified by using primers conp28-f1 , conp28-r1 , conp28-f2 , and conp28-r2 , as described ( 3,9 ) .
amplicons of p44/msp2 were subjected to ta cloning ( ta cloning kit ; life technologies , grand island , ny , usa ) , and randomly selected recombinant clones were sequenced and analyzed phylogenetically ( figure 1 ) .
a total of 28 p44/msp2 clones from case - patient 1 shared 27.5%100% similarity with each other and were widely dispersed in the tree .
the 40 clone sequences from case - patient 2 shared 97.5%100% similarity with each other and grouped into a single cluster . using blast ( http://blast.ncbi.nlm.nih.gov ) , we compared the sequences with those in genbank ; 27 previously identified p44/msp2 variants from human isolates and ticks collected in japan were identified as the closest relatives to p44/msp2 cloned from the 2 patients .
we included the 27 variants in the tree ; however , some were widely separated from the related clones ( figure 1 ) .
for case - patient 2 , the 389-bp sequence of the 16s rdna amplicon ( determined by direct sequencing ) was 100% identical to that of r. japonica yh ( genbank accession no .
phylogenetic analysis of anaplasma phagocytophilum p44/msp2 multigenes detected in blood from 2 men in kochi prefecture , japan .
each p44/msp2 pcr product was cloned ( ta cloning kit ; life technologies , grand island , ny , usa ) into the pcr2.1 vector , after which recombinant clones were randomly selected and the dna inserts were sequenced .
the tree was constructed on the basis of the 117133 aa sequences of the p44/msp2 genes by using the neighbor - joining method . the closest relatives to sequences for the 2 case - patients
those sequences have been published in genbank : patient2-day27 ( obtained from a us patient ) ; p44 - 2 , p44 - 8 , p44 - 10 , p44 - 11 , p44 - 13 , p44 - 18e , p44 - 28 , p44 - 35 , p44 - 39 , p44 - 40 , p44 - 41 , p44 - 48 , varhh2 , and wmsp5 are from human isolates ; and 44-kda outer membrane proteins are from ticks collected in japan .
boldface font indicates the 28 p44/msp2 genes from case - patient 1 and the 40 from case - patient 2 .
data in parentheses indicate the number of p44/msp2 clones with identical sequences and the sequence accession numbers .  
serologic evidence of infection was demonstrated by using indirect immunofluorescence assay ( ifa ) and western blot analysis as described ( 10,11 ) . in ifas , igm and/or igg from serum samples from
the case - patients reacted with a. phagocytophilum cultured in thp-1 rather than hl60 cells , and seroconversion was stronger in convalescent - phase serum samples ( table 2 ) .
igg titers against r. japonica were also higher in convalescent - phase samples from case - patient 2 .
western blot analysis further confirmed the specific reaction to the 44-kda outer membrane proteins ( p44s ) of a. phagocytophilum cultured in thp-1 cells and/or to the recombinant p44 - 1 ( rp44 - 1 ) in serum samples ( figure 2 , table 2 ) .
however , using the same serum samples , we could not detect p44 antigens of a. phagocytophilum propagated in hl60 cells ( data not shown ) , supporting the ifa result . *
all western blot testing using recombinant p44 - 1 antigen detected igm and igg ; the antigen reacted with all sera tested , as shown in figure 2 . 
western blot analyses , using recombinant p44 - 1 protein ( rp44 - 1 ) and anaplasma phagocytophilum 
infected thp-1 cells as antigens , of serum samples from 2 men , case - patients 1 and 2 , who had a. phagocytophilum infection , kochi prefecture , japan .
the escherichia coli , which produced rp44 - 1 , was kindly provided by yasuko rikihisa ( ohio state university , columbus , oh , usa ) .
the rp44 - 1 and the rabbit hyperimmune serum ( positive control serum ) were prepared as described ( 11,12 ) .
the primary human serum samples tested were diluted 200- to 400-fold ; rabbit serum sample ( positive control ) was diluted 2,000-fold .
the goat antihuman igg and igm alkaline phosphatase conjugates ( life technologies , grand island , ny , usa ) were used as secondary antibodies .
rp , rp44 - 1 antigen ; in , infected thp-1 ; un , uninfected thp-1 cells . in central and western japan , most cases of tickborne infectious and febrile disease have been reported as jsf ( 1,13 ) , and r. japonica has been frequently detected in ixodid ticks in these areas .
we found a. phagocytophilum infection in several species of ticks , and at least 3 species ( h. formosensis , h. longicornis , and i. ovatus ) seem to be associated with r. japonica and a. phagocytophilum ( 7,8 ) .
national surveillance during 19992010 , showed that jsf was endemic in kochi prefecture during 19992004 .
more recently , jsf - endemic areas are mie , kagoshima , wakayama , and kumamoto prefectures rather than kochi prefecture .
our survey demonstrating the presence of a. phagocytophilum  infected ticks in mie and kagoshima prefectures ( 8) indicates that there is a risk for dual infection with r. japonica and a. phagocytophilum in jsf - endemic areas of japan .
a. phagocytophilum cultured in hl60 cells is generally used as a source of antigen for serodiagnosis of human anaplasmosis .
our findings show , however , that titers of antibody against a. phagocytophilum propagated in thp-1 cells were higher than those propagated in hl60 cells .
we further analyzed the transcription of p44/msp2 multigenes encoding p44 repertoires ( major antigens of a. phagocytophilum ) in infected hl60 and thp-1 cells by using reverse transcription pcr followed by ta cloning as described ( 7 ) .
the analyses showed that a transcript from the p44 - 60 gene and another from the p44 - 47 gene ( 75% and 25% of transcripts tested , respectively ) were dominantly expressed in a. phagocytophilum propagated in thp-1 cells but not in hl60 cells ; several transcript species other than p44 - 60 and p44 - 47 of p44/msp2 multigenes were expressed in a. phagocytophilum propagated in hl60 cells ( data not shown ) .
a previous proteomic study supported the variety of p44 repertoires produced by a. phagocytophilum in hl60 cells ( 14 ) .
the difference of p44/msp2 expression between hl60 and thp-1 cell cultures may reflect the discrepancy of antibody titers obtained by ifas .
furthermore , in ifas using infected thp-1 antigens , igm titers tended to be higher than igg titers , even in convalescent - phase serum samples .
these patients probably produced igg reactive with p44 species other than p44 - 60 and p44 - 47 that were dominantly expressed in a. phagocytophilum propagated in thp-1 cells ; western blot analysis showed that igg in patients strongly bound to recombinant p44 - 1 rather than p44s ( probably including p44 - 60 and p44 - 47 ) of a. phagocytophilum propagated in thp-1 cells .
thus , cases of human anaplasmosis could go undiagnosed if only infected hl60 cells , and not thp-1 cells , are used as antigen for serodiagnosis of rickettsiosis - like infections , as is currently done when using ifas .
we documented 2 cases of human granulocytic anaplasmosis in japan , 1 with and 1 without jsf coinfection . to avoid misdiagnosing cases of human anaplasmosis
, we recommend that a. phagocytophilum propagated in thp-1 and in hl60 cells be used as antigens for the serodiagnosis of rickettsiosis - like infections .
we retrospectively confirmed 2 cases of human anaplasma phagocytophilum infection . 
 patient blood samples contained unique p44/msp2 for the pathogen , and antibodies bound to a. phagocytophilum antigens propagated in thp-1 rather than hl60 cells . 
 unless both cell lines are used for serodiagnosis of rickettsiosis - like infections , cases of human granulocytic anaplasmosis could go undetected .