with approval from the oregon state university animal care and use committee ( animal care and use proposal no .
4329 ) , blood samples were collected from 92 horses being evaluated for medical conditions ( e.g. , anorexia , weight loss , lameness , administration of endoparasiticides and ectoparasiticides ) or before elective surgery ( e.g. , castration , wound repair ) at the clinic in mrida from august 28 through september 4 , 2013 .
( a. phagocytophilum and a. platys ) , b. burgdorferi sensu stricto , and ehrlichia spp .
( e.canis , e. chaffeensis , and e. ewingii ) by using the elisa - based assay snap 4dxplus ( idexx laboratories , inc . ,
westbrook , me , usa ) according to the manufacturer s instructions ( 9 ) .
the assay does not use a host species  specific conjugate and can therefore be used in research to screen mammals other than dogs . according to assay results , 51 ( 55% ) horse serum samples
the 51 ehrlichia spp.reactive serum samples were subsequently stored at 28c for up to 1 week during transport to the united states . for pcr testing , samples were shipped ( us department of agriculture import permit no .
13846 ) to the intracellular pathogens research laboratory , center for comparative medicine and translational research , north carolina state university , college of veterinary medicine .
dna extraction from 200 l of edta - anticoagulated whole blood was performed by using a qiasymphony dna mini kit ( qiagen , valencia , ca , usa ; catalog no .
previously described pcrs were used to amplify a 420-bp fragment of the 16s rrna gene , a 620-bp fragment of the groel gene , and a 300-bp fragment of the ehrlichia spp .
a larger , 600-bp , fragment of the sodb gene was amplified from 1 sample that was positive by pcr by using the following unpublished primers : sodbehrl600-f 5-atgtttactttacctgaacttccatatc-3 and sodbehrl600-r 5-atctttgagctgcaaaatcccaatt-3. positive ( anaplasma or ehrlichia spp . plasmid dna ) and negative ( rnase - free molecular - grade water and a dna extraction control consisting of uninfected canine genomic dna ) controls were used for each assay .
( research triangle park , nc , usa ) , and alignments were compared with those of genbank sequences by using alignx software ( vector nti advance version 11.5 ; invitrogen , carlsbad , ca , usa ) .
of the 51 samples tested by 16s rdna pcr , the rdna amplicon sequences were identical for 4 ( 8% ) .
* sequences for partial 16s rrna , groel , and sodb genes are compared with genbank database sequences from ehrlichia ruminantium , e. canis , e. chaffeensis , e. ewingii , anaplasma marginale , a. phagocytophilum , and neorickettsia risticii . 
genbank sequence accession numbers for 16s rrna , groel , and sodb for ehrlichia sp . from horses in nicaragua are kj434178 , kj434179 , and kj434180 , respectively .
according to serologic , pcr amplification , and dna sequencing results , tick - infested horses in mrida , nicaragua , might be infected with a potentially novel ehrlichia species .
initial serologic screening with the rapid elisa indicated that exposure to > 1 ehrlichia species is common among horses in nicaragua ( 55% ) .
for dogs , snap 4dxplus results can be positive after exposure to e. canis , e. chaffeensis , e. ewingii , and potentially e. muris and panola mountain ehrlichiae ( 2,3,6 ) . before this study , equine exposure to a. phagocytophilum ( previous designation e. equi ) in nicaragua
was considered more likely because equine exposure has occurred in guatemala ; this rickettsial organism is pathogenic for cats , dogs , horses , and humans ( 12 ) .
however , in north america , a. phagocytophilum is transmitted by i. scapularis and i. pacificus ticks , which have not been reported in nicaragua . in future studies of horses in nicaragua , ticks will be collected for identification . in guatemala ,
r. microplus and a. cajennense ticks were the predominant species found on cattle , whereas d. nitens and a. cajennense ticks were most commonly found on horses ( 12 ) .
of note , in guatemala , tick infestation levels were substantially higher and body condition scores lower for horses than for cattle .
also , cattle were exposed to an agent with serologic cross - reactivity and close genetic relatedness to e. ruminantium .
the partial 16s rdna sequences obtained from these horses most likely represent a novel species of ehrlichia .
this conclusion is further supported by sequence analysis of 2 protein - coding genes , sodb and groel . partial sequences from sodb and groel genes demonstrated similarity to ehrlichia spp .
sequences found in genbank , but they were not 100% identical to any sequences deposited to date .
recommend that gene homology for organisms of identical species and genus be 99.8% and 98.1% , respectively ( 13 ) .
although identical species are typically defined as being > 99% identical with a reference sequence , the percentage identity needed to define a separate species is debated , ranging from 97% to 99.5% ( 14 ) .
calculation of values can be based on alignment methods , reference databases , and number of basepairs in the sequence .
recommend that for novel species identification and rickettsiae classification , protein - coding genes should be used , specifically the 4 protein - coding genes glta , ompa , ompb , and gene d in addition to the 16s rrna gene . to date , 16s rdna sequences or whole - genome sequencing have been used to classify ehrlichia species and strains . collectively , and as depicted in the figure ( in which phylogenetic alignment trees for all 3 genes tested in this study were constructed by using reference sequences from representative members of the genera anaplasma , ehrlichia , and neorickettsia ) , the genetic findings in this study support infection of horses in nicaragua with a novel ehrlichia species .
to confirm this possibility , future efforts will focus on cell culture isolation of the ehrlichia organism from horses in nicaragua .
phylogenetic trees of ehrlichia sp . from horses in nicaragua and selected bacterial species ( genbank accession numbers for reference sequences in parenthesis ) based on partial sequences from genes coding for 16srrna ( a ) , groel ( b ) , and sodb ( c ) .
sequences were aligned by using muscle version 3.7 ( http://www.ebi.ac.uk/tools/msa/muscle/ ) , and alignments were refined by using gblocks version 0.91b ( http://www.idtdna.com/gblocks.com ) .
phylogenetic trees were constructed by using phyml version 3.0 alrt ( http://code.google.com/p/phyml/ ) under the hky85 model , and the resulting trees were rendered by using treedyn version 198.3 ( http://www.treedyn.org/ ) .
scale bars indicate number of substitutions per site , and the numbers in the branches represent percentage support of the node .
at least 4 ehrlichia species have been implicated as being pathogenic for canids and humans ( 13 ) .
thus , future studies should also determine whether dogs , other animals , and humans in nicaragua are exposed to and infected with this potentially novel ehrlichia species .
 ehrlichia sp . dna was amplified from 4 ehrlichia - seroreactive horses from mrida , nicaragua . 
 sequencing of 16s rdna , sodb , and groel genes indicated that the bacterium is most likely a novel ehrlichia species . 
 the tick vector and the potential for canine and human infection remain unknown . 
The Study
Conclusions