BACKGROUND
the genus anaplasma  contains obligate intracellular organisms found exclusively within membrane-bound inclusions or vacuoles in the cytoplasm of both vertebrate and invertebrate host cells  <cit> .
this genus includes pathogens of ruminants, a. marginale, a. centrale, a. bovis , and a. ovis.
also included in this genus is a. phagocytophilum  agent), which infects a wide range of hosts including humans and wild and domesticated animals, and a. platys  which is infective for dogs.
in dogs, a. platys develops within platelets and is the etiologic agent of canine infectious cyclic thrombocytopenia, but infected dogs are usually asymptomatic  <cit> .
canine infections of a. platys have been reported throughout the world, including the united sates  <cit> , spain  <cit> , france  <cit> , greece  <cit> , italy  <cit> , taiwan  <cit> , china  <cit> , thailand  <cit> , japan  <cit> , venezuela  <cit> , and australia  <cit> .
however, a. platys infection is difficult to detect in vivo because the bacteremias are usually low  <cit> .
furthermore, serologic tests may be inaccurate because they are cross-reactive with other anaplasma  <cit> .
recently, a pcr assay was optimized to allow for accurate identification of a. platys infection in dogs  <cit> .
the pcr test, confirmed by sequence analysis of amplicons, is considered to be the most reliable diagnostic test for a. platys to date.
despite the worldwide distribution of a. platys, limited information is available on the genetic diversity of a. platys strains  <cit> .
herein, we characterized strains of a. platys from dogs in palermo, sicily, italy, using a combination of pcr and sequence analysis of 16s rdna, heat shock operon groesl and the citrate synthase  genes.
methods
blood samples
blood was collected from  <dig> dogs  during 2003– <dig> in the province of palermo, sicily, italy, for these studies.
blood was collected into sterile tubes with anticoagulant , held at 4°c until arrival at the laboratory and then stored at -20°c for dna extraction.
dna extraction, pcr and sequence analysis
dna was extracted from blood and tick samples using the genelute mammalian genomic dna miniprep kit .
the a. marginale/a.
centrale/a.
ovis and a. phagocytophilum msp <dig> genes were amplified by pcr as reported previously  <cit> .
the anaplasma spp.
16s rdna was amplified by pcr using oligonucleotide primers 16sana-f  and 16sana-r  as described previously  <cit> .
the a. platys-specific 16s rdna, groesl and glta pcrs were done as reported by martin et al.
<cit>  and inokuma et al.
<cit> , respectively.
pcr reactions contained  <dig> μl  dna and  <dig> pmol of each primer in a 50-μl volume  employing the access rt-pcr system .
reactions were performed in an automated dna thermal cycler  for  <dig> cycles.
control reactions were done using the same procedures and reagents described above but without dna added to the pcr reaction to rule out pcr contaminations.
carrying-over was ruled out due to the low number of pcr positive samples and the differences in the amplicon sequences.
pcr products were electrophoresed on 1% agarose gels to check the size of amplified fragments by comparison to a dna molecular weight marker .
amplified 16s rdna, groesl and glta fragments were resin purified  and cloned into pgem-t vector  for sequencing both strands by double-stranded dye-termination cycle sequencing .
at least two independent clones were sequenced.
multiple sequence alignment was performed using the program alignx  with an engine based on the clustal w algorithm  <cit> .
blast  <cit>  was used to search the ncbi databases to identify previously reported sequences with identity to those obtained in the study described herein.
sequence accession numbers
the genbank accession numbers for glta sequences of a. platys strains are .
RESULTS
prevalence of a. platys in dogs from sicily
the observed prevalence of anaplasma spp.
was analyzed by pcr and sequence analysis of 16s rdna amplicons.
of the  <dig> dogs analyzed,  <dig>  were positive for anaplasma spp.
dna .
sequence analysis of 16s rdna amplicons resulted in 100% identity to previously reported a. platys sequences.
none of the samples were positive for a. marginale, a. centrale, a. ovis and a. phagocytophilum dna.
previous 16s rdna pcr-based a. platys studies in dogs reported observed prevalences of 33% , 32% , 45%  and 16% .
the analysis reported herein included a greater number of samples but the observed prevalence of a. platys in dogs was lower than in previous studies.
these results suggested that a. platys infection in dogs in sicily may be very low.
however, differences in the sensitivity of the pcr due to the size of the amplicon and primer sequences may affect the results of prevalence studies reported by different groups.
molecular characterization of a. platys strains from sicily
the  <dig> positive dog samples were characterized with a. platys-specific 16s rdna, groesl and glta pcr and sequence analysis.
all dogs had 16s rdna and groesl sequences identical to  and  a. platys spanish and italian strain sequences reported previously, respectively.
these results agree with previous reports in which little genetic diversity was observed between 16s rdna and groesl sequences of a. platys strains  <cit> .
the sequence of a. platys glta resulted in  <dig> different genotypes .
the sequences of a. platys from samples miky, dog  <dig> and dog  <dig> were present in 9/ <dig>  3/ <dig> and 2/ <dig> of the positive dog samples, respectively.
a single nucleotide change in dog  <dig> sequence resulted in an amino acid change .
although the information about a. platys sequences is limited, these results suggest that glta sequences may be more diverse than 16s rdna and groesl sequences.
the result of glta sequence analysis suggested that at least three different genotypes of a. platys infect dogs in sicily.
two of the glta sequences of sicilian a. platys strains  were different from sequences reported previously .
however, the glta sequence of miky strain was identical to the sequence of the sommieres french strain.
huang et al.
<cit>  suggested that a. platys strains are not geographically segregated.
although limited by the number of sequences available, the results of our study suggested that glta may provide some phylogeographic information about a. platys strains.
nevertheless, although 16s rdna, groesl and glta sequences may be useful for phylogenetic studies of anaplasma spp.
<cit> , they were not informative for phylogenetic studies of a. platys strains.
the genetic diversity of a. marginale, a. phagocytophilum and a. ovis strains have been documented using different genetic markers  <cit> .
the results reported herein suggested that the genetic variation in a. platys may be similar to a. ovis but less than that observed in a. marginale and a. phagocytophilum.
this low genetic variation may have resulted from restricted movement of infected hosts compared to a. marginale-infected cattle and/or the limited host range of a. ovis and a. platys as compared with a. phagocytophilum  <cit> .
the tick vectors for the transmission of a. platys have not been extensively characterized.
rhipicephalus sanguineus  <cit> , dermacentor auratus  <cit>  and hyalomma truncatum  <cit>  have been suggested as possible vectors of a. platys.
however, experimental transmission of a. platys with these tick species has not been demonstrated  <cit> .
nevertheless, ticks of the genera rhipicephalus, dermacentor and hyalomma are present in sicily and could act as vectors of a. platys in this region  <cit> .
CONCLUSIONS
low observed prevalence  of a. platys was detected in dogs from sicily by pcr and sequence analysis of 16s rdna, groesl and glta genes.
three different glta genotypes of a. platys were identified in these dogs.
the results reported herein suggested that genetic diversity of a. platys strains may be similar to a. ovis but lower than that for a. marginale and a. phagocytophilum.
the lower genetic diversity of a. platys may have resulted from restricted movement of infected hosts as compared to a. marginale-infected cattle and/or the limited host range of a. ovis and a. platys as compared with a. phagocytophilum.
these results expand our knowledge about a. platys and encourage further research to characterize genetic diversity of a. platys strains worldwide.
competing interests
the author declare that they have no competing interests.
authors' contributions
josé de la fuente has designed and performed the sequence analyses and wrote the manuscript.
alessandra torina has organized and supervised the initial screening of dog samples by pcr.
victoria naranjo, silviane nicosia, angelina alongi and francesco la mantia have performed all the dna extractions and pcr analyses.
katherine m. kocan has contributed to the final draft of the manuscript.
all authors have read and approved the final manuscript.

 the genus anaplasma contains obligate intracellular organisms found exclusively within membrane-bound inclusions or vacuoles in the cytoplasm of both vertebrate and invertebrate host cells. this genus includes pathogens of ruminants, a. marginale, a. centrale, a. bovis, and a. ovis. also included in this genus is a. phagocytophilum agent), which infects a wide range of hosts including humans and wild and domesticated animals, and a. platys which is infective for dogs. in dogs, a. platys develops within platelets and is the etiologic agent of canine infectious cyclic thrombocytopenia, but infected dogs are usually asymptomatic. canine infections of a. platys have been reported throughout the world, including the united sates, spain, france, greece, italy, taiwan, china, thailand, japan, venezuela, and australia. however, a. platys infection is difficult to detect in vivo because the bacteremias are usually low. furthermore, serologic tests may be inaccurate because they are cross-reactive with other anaplasma. recently, a pcr assay was optimized to allow for accurate identification of a. platys infection in dogs. the pcr test, confirmed by sequence analysis of amplicons, is considered to be the most reliable diagnostic test for a. platys to date. despite the worldwide distribution of a. platys, limited information is available on the genetic diversity of a. platys strains. herein, we characterized strains of a. platys from dogs in palermo, sicily, italy, using a combination of pcr and sequence analysis of 16s rdna, heat shock operon groesl and the citrate synthase genes. methods blood samples blood was collected from dogs during 2003– in the province of palermo, sicily, italy, for these studies. blood was collected into sterile tubes with anticoagulant, held at 4°c until arrival at the laboratory and then stored at -20°c for dna extraction. dna extraction, pcr and sequence analysis dna was extracted from blood and tick samples using the genelute mammalian genomic dna miniprep kit. the a. marginale/a. centrale/a. ovis and a. phagocytophilum msp genes were amplified by pcr as reported previously. the anaplasma spp. 16s rdna was amplified by pcr using oligonucleotide primers 16sana-f and 16sana-r as described previously. the a. platys-specific 16s rdna, groesl and glta pcrs were done as reported by martin et al. and inokuma et al., respectively. pcr reactions contained μl dna and pmol of each primer in a 50-μl volume employing the access rt-pcr system. reactions were performed in an automated dna thermal cycler for cycles. control reactions were done using the same procedures and reagents described above but without dna added to the pcr reaction to rule out pcr contaminations. carrying-over was ruled out due to the low number of pcr positive samples and the differences in the amplicon sequences. pcr products were electrophoresed on 1% agarose gels to check the size of amplified fragments by comparison to a dna molecular weight marker. amplified 16s rdna, groesl and glta fragments were resin purified and cloned into pgem-t vector for sequencing both strands by double-stranded dye-termination cycle sequencing. at least two independent clones were sequenced. multiple sequence alignment was performed using the program alignx with an engine based on the clustal w algorithm. blast was used to search the ncbi databases to identify previously reported sequences with identity to those obtained in the study described herein. sequence accession numbers the genbank accession numbers for glta sequences of a. platys strains are.  prevalence of a. platys in dogs from sicily the observed prevalence of anaplasma spp. was analyzed by pcr and sequence analysis of 16s rdna amplicons. of the dogs analyzed, were positive for anaplasma spp. dna. sequence analysis of 16s rdna amplicons resulted in 100% identity to previously reported a. platys sequences. none of the samples were positive for a. marginale, a. centrale, a. ovis and a. phagocytophilum dna. previous 16s rdna pcr-based a. platys studies in dogs reported observed prevalences of 33%, 32%, 45% and 16%. the analysis reported herein included a greater number of samples but the observed prevalence of a. platys in dogs was lower than in previous studies. these results suggested that a. platys infection in dogs in sicily may be very low. however, differences in the sensitivity of the pcr due to the size of the amplicon and primer sequences may affect the results of prevalence studies reported by different groups. molecular characterization of a. platys strains from sicily the positive dog samples were characterized with a. platys-specific 16s rdna, groesl and glta pcr and sequence analysis. all dogs had 16s rdna and groesl sequences identical to and a. platys spanish and italian strain sequences reported previously, respectively. these results agree with previous reports in which little genetic diversity was observed between 16s rdna and groesl sequences of a. platys strains. the sequence of a. platys glta resulted in different genotypes. the sequences of a. platys from samples miky, dog and dog were present in 9/ 3/ and 2/ of the positive dog samples, respectively. a single nucleotide change in dog sequence resulted in an amino acid change. although the information about a. platys sequences is limited, these results suggest that glta sequences may be more diverse than 16s rdna and groesl sequences. the result of glta sequence analysis suggested that at least three different genotypes of a. platys infect dogs in sicily. two of the glta sequences of sicilian a. platys strains were different from sequences reported previously. however, the glta sequence of miky strain was identical to the sequence of the sommieres french strain. huang et al. suggested that a. platys strains are not geographically segregated. although limited by the number of sequences available, the results of our study suggested that glta may provide some phylogeographic information about a. platys strains. nevertheless, although 16s rdna, groesl and glta sequences may be useful for phylogenetic studies of anaplasma spp., they were not informative for phylogenetic studies of a. platys strains. the genetic diversity of a. marginale, a. phagocytophilum and a. ovis strains have been documented using different genetic markers. the results reported herein suggested that the genetic variation in a. platys may be similar to a. ovis but less than that observed in a. marginale and a. phagocytophilum. this low genetic variation may have resulted from restricted movement of infected hosts compared to a. marginale-infected cattle and/or the limited host range of a. ovis and a. platys as compared with a. phagocytophilum. the tick vectors for the transmission of a. platys have not been extensively characterized. rhipicephalus sanguineus, dermacentor auratus and hyalomma truncatum have been suggested as possible vectors of a. platys. however, experimental transmission of a. platys with these tick species has not been demonstrated. nevertheless, ticks of the genera rhipicephalus, dermacentor and hyalomma are present in sicily and could act as vectors of a. platys in this region.  low observed prevalence of a. platys was detected in dogs from sicily by pcr and sequence analysis of 16s rdna, groesl and glta genes. three different glta genotypes of a. platys were identified in these dogs. the results reported herein suggested that genetic diversity of a. platys strains may be similar to a. ovis but lower than that for a. marginale and a. phagocytophilum. the lower genetic diversity of a. platys may have resulted from restricted movement of infected hosts as compared to a. marginale-infected cattle and/or the limited host range of a. ovis and a. platys as compared with a. phagocytophilum. these results expand our knowledge about a. platys and encourage further research to characterize genetic diversity of a. platys strains worldwide. competing interests the author declare that they have no competing interests. authors' contributions josé de la fuente has designed and performed the sequence analyses and wrote the manuscript. alessandra torina has organized and supervised the initial screening of dog samples by pcr. victoria naranjo, silviane nicosia, angelina alongi and francesco la mantia have performed all the dna extractions and pcr analyses. katherine m. kocan has contributed to the final draft of the manuscript. all authors have read and approved the final manuscript. 
the prevalence of a. platys in dogs in sicily, as demonstrated by pcr and sequence analysis of the 16s rdna, groesl and glta genes, was 4%. at least three different strains of a. platys were identified in dogs from sicily by pcr and sequence analyses of the 16s rdna, groesl and glta genes. the present study was designed to characterize a. patys strains in dogs from palermo, sicily, italy, using a combination of pcr and sequence analysis of the 16s rdna, heat shock operon groesl and citrate synthase genes. however, one of the glta, 16s rdna and groesl sequences were identical to the sequence of a. platys strains from other regions of the world characterized previously. the genetic diversity of anaplasma platys strains is currently poorly defined. the results reported herein suggested that genetic diversity of a. platys strains may be similar to a. ovis, but lower than the diversity reported for a. marginale and a. phagocytophilum. this lower genetic diversity may have resulted from restricted movement of infected hosts compared to a. marginale-infected cattle and/or the limited host range of a. ovis and a. platys as compared with a. phagocytophilum. two of the glta sequences of sicilian a. platys strains were different from sequences reported previously. these results expand our knowledge about a. platys and encourage further research for analysis of the genetic variation of a. platys strains worldwide. none of the samples were positive for a. marginale, a. centrale, a. ovis and a. phagocytophilum dna. three different glta genotypes of a. platys were identified in dogs from sicily.
 the genetic diversity of anaplasma platys strains is currently poorly defined. the present study was designed to characterize a. patys strains in dogs from palermo, sicily, italy, using a combination of pcr and sequence analysis of the 16s rdna, heat shock operon groesl and citrate synthase genes.  blood was collected from dogs during 2003– in the province of palermo, sicily, italy. the prevalence of a. platys in dogs in sicily, as demonstrated by pcr and sequence analysis of the 16s rdna, groesl and glta genes, was 4%. none of the samples were positive for a. marginale, a. centrale, a. ovis and a. phagocytophilum dna. three different glta genotypes of a. platys were identified in dogs from sicily. two of the glta sequences of sicilian a. platys strains were different from sequences reported previously. however, one of the glta, 16s rdna and groesl sequences were identical to the sequence of a. platys strains from other regions of the world characterized previously.  at least three different strains of a. platys were identified in dogs from sicily by pcr and sequence analyses of the 16s rdna, groesl and glta genes. the results reported herein suggested that genetic diversity of a. platys strains may be similar to a. ovis, but lower than the diversity reported for a. marginale and a. phagocytophilum. this lower genetic diversity may have resulted from restricted movement of infected hosts compared to a. marginale-infected cattle and/or the limited host range of a. ovis and a. platys as compared with a. phagocytophilum. these results expand our knowledge about a. platys and encourage further research for analysis of the genetic variation of a. platys strains worldwide. 