we conducted real - time pcr analyses targeting the msp2 gene on genomic ( g ) dna of 12,606 i. scapularis ticks collected across canada ( technical appendix 1 ) .
169 ( 1.3% ) ticks were pcr - positive ( table 1 ) . there were significant differences ( 5  =  129.7 , p<0.001 ) in the proportions of ticks infected with a. phagocytophilum among geographic regions ; with a greater proportion of pcr - positive ticks in manitoba than in ontario , quebec , and the atlantic provinces . * data from these provinces were not included in the statistical analyses because sample sizes ( 20072010 ) were < 100 ticks .
we developed pcr - based assays to distinguish the ap - ha strain from the ap - variant 1 strain of a. phagocytophilum based on dna sequence comparisons of the 16s rrna gene of each strain ( technical appendix 1 ) over a much larger region ( 875 bp ) than in previous studies ( 1,3 ) .
in addition to the 2 nucleotide differences described previously ( 1,3 ) , a third difference ( at position 536 ) was detected in the aligned sequences ( technical appendix 2 ) .
this nucleotide alteration in the dna sequence of the ap - variant 1 strain was associated with a restriction site for the endonuclease kpn2i ( t / ccgga ) that was absent in the sequence of the ap - ha strain .
two pcr - rflp assays ( technical appendix 1 ) , developed at different laboratories , were designed on the basis of this sequence difference and tested on 22 amplicons derived from a. phagocytophilum  infected ticks collected in minnesota , usa
eighteen amplicons remained undigested ( i.e. , a single band of 920 bp ) , and 3 amplicons had 2 bands ( 360 and 550 bp ) , representing the expected patterns for the ap - ha strain and ap - variant 1 strain , respectively ( figure 1 ) .
these results were confirmed on the basis of comparisons of the dna sequences of representative samples . the rflp pattern of 1 amplicon , derived from the gdna of a female tick from itasca state park , minnesota , consisted of 3 bands ( 360 , 550 and 920 bp ) ( technical appendix 2 ) , suggesting a mixture of the 2 strains .
additional analyses of another 125 amplicons from a. phagocytophilum - infected ticks revealed that 79 ( 63% ) had rflp patterns consistent with the ap - variant 1 strain , and 46 ( 37% ) had rflp patterns of the ap - ha strain .
the dna sequences of a subset of these samples ( n = 58 ) showed 100% agreement between rflp pattern and strain type of a. phagocytophilum ( i.e. , 24 of the ap - ha strain and 34 of the ap - variant 1 strain ) .
restriction fragment length polymorphism patterns of 16s dna for 7 anaplasma phagocytophilum pcr  positive ixodes scapularis ticks .
all amplicons were produced by semi - nested pcr and digested with the restriction enzyme kpn2i .
we also designed a custom taqman snp genotyping assay ( https://www.lifetechnologies.com/order/custom-genomic-products/tools/genotyping ) to differentiate the two a. phagocytophilum strains on the basis of a nucleotide difference at the 5 end of the 16s rrna gene sequence ( technical appendix 1 ) .
the snp assay clearly discriminated between ticks infected with the ap - ha and/or ap - variant 1 strains ( figure 2 ) .
of the 142 amplicons tested by this assay , 82 ( 58% ) contained the ap - variant 1 strain , 59 ( 42% ) contained the ap - ha strain , and 1 contained a mixture of both strains , which was in 100% agreement with the results of the rflp analyses and dna sequencing .
the results of the snp analyses also revealed a significant difference ( 2  =  40.48 , p<0.001 ) in the proportions of i. scapularis ticks infected with the ap - ha strain among geographic regions ( table 2 ) . a smaller proportion of ticks from central provinces were infected with the ap - ha strain when compared with those from the prairie and atlantic provinces .
allelic discrimination plot for the anaplasma single nucleotide polymorphism assay based on the 16s rna gene .
blue circles represent samples that contain the ap - ha strain ; green circles represent samples that contain the ap - variant 1 strain .
includes 13 ticks listed in table 1 and an additional 11 a. phagocytophilum  infected ticks collected by drag sampling at 2 sites in ontario .
a small proportion ( 1.3% ) of i. scapularis ticks collected in canada were infected with a. phagocytophilum .
the prevalence of a. phagocytophilum-infected ticks differed among geographic regions , but the potential significance of this finding needs to be explored further . although knowledge of the prevalence of a. phagocytophilum  infected i. scapularis ticks provides some information for determining the public health risks for hga , the prevalence of the ap - ha strain of a. phagocytophilum in blacklegged ticks should be considered for risk assessment , since the ap - ha strain , and not the ap - variant 1 strain , is most often associated with clinical cases of hga ( 4 ) . for the current study
, 3 pcr - based assays were developed to distinguish the ap - ha strain from the ap - variant 1 strain .
each of the 3 assays detected the presence of both strains in the gdna of a female tick .
mixed infections of both strains in i. scapularis ticks have been reported ( 4 ) , but appear to be uncommon .
results of these assays also were 100% concordant with the results of 2 rflp assays ( developed at different laboratories ) that were tested on the same gdna samples .
similarly , there was total concordance in the identifications of the a. phagocytophilum strains present within 125 infected ticks collected across canada by using the seminested pcr - rflp assay and the taqman snp genotyping assay .
the results of these analyses revealed significant differences in the proportion of blacklegged ticks infected with the ap - ha strain among geographic regions ( p<0.001 ) .
the public health implications of these findings need to be examined in more detail , using the molecular assays developed in this study .
the taqman snp genotyping assay is ideal for clinical and epidemiologic use where it may be essential to distinguish between the 2 strains of a. phagocytophilum in i. scapularis to assess the potential risk for human infection .
however , in a clinical setting , it remains to be established how this assay would be incorporated into or supplement the current diagnostic approach for detecting a. phagocytophilum infections in humans .
this test is less technically demanding and takes less time to perform than nested / seminested pcr - rflps and dna sequencing analyses . moreover , this method eliminates the need for postamplification manipulations and technical problems that are sometimes associated with rflp analyses of amplicons produced by nested pcr ( 14,15 ) .
nonetheless , given the 100% concordance in the results of the different analytical methods , the pcr - rflp assays provide a reliable and cost - effective approach for distinguishing the ap - ha strain from the ap - variant 1 strain of a. phagocytophilum .
the pcr - rflp assays will be particularly useful in research laboratories that lack the capacity to conduct real - time pcrs providing an independent and relatively inexpensive method to confirm the results of the snp assay .
samples , dna extraction and prevalence of a. phagocytophilum in i. scapularis genetic test results for a. phagocytophilum
 we developed pcr - based assays to distinguish a human pathogenic strain of anaplasma phagocytophilum , ap - ha , from ap - variant 1 , a strain not associated with human infection . 
 the assays were validated on a. phagocytophilum - infected blacklegged ticks ( ixodes scapularis ) collected in canada . 
 the relative prevalence of these 2 strains in i. scapularis ticks differed among geographic regions . 
The Study
Conclusions
None