A 16S rRNA-based PCR assay for detection and identification of granulocytic Ehrlichia species in dogs, horses, and cattle

Abstract

A PCR-based assay was developed for detecting DNA of granulocytic ehrlichiae in blood samples from dogs, horses, and cattle. Primers were designed from 16S rRNA sequence information to specifically amplify DNA from a newly identified Swedish Ehrlichia species. The 16S rRNA nucleotide sequence of this Swedish species differs in only two and three positions from the sequences of Ehrlichia phagocytophila and Ehrlichia equi, respectively, which were also amplified by this PCR system. For evaluation, PCR results were compared with microscopic examination of stained blood smears for the detection of granulocytes containing ehrlichiae (morulae). Thirty-four of 36 microscopically positive samples were also positive by PCR, and 6 microscopically negative samples were negative by PCR as well. Six samples, in which morulae-like structures had been seen, were negative by PCR, also at a lower annealing temperature and when a reamplification of the first PCR products was performed. The identities of the PCR products from some canine and equine isolates were verified by direct DNA sequencing and were found to be identical with the Ehrlichia sequence found in these animal species that had been obtained earlier. The sequences of a segment of approximately 600 nucleotides from two bovine isolates were identical to that of E. phagocytophila, whereas the sequence of another bovine isolate differed in two positions from that of E. phagocytophila and in three positions from the sequences of the canine and equine isolates. Serum samples were analyzed by indirect fluorescent-antibody testing. Seventy-three percent of the animals which were positive by microscopy and PCR also had positive antibody titers. However, it was not possible to rely on a single serological result for diagnosis of present infection. It was, therefore, concluded that PCR was the most reliable method, useful in the clinical laboratory for specific and early diagnosis of granulocytic ehrlichiosis in animals.

Acute Clinical, Hematologic, Serologic, and Polymerase Chain Reaction Findings in Horses Experimentally Infected with a European Strain of Anaplasma phagocytophilum

“…Sequences for each gene were identical between the sampled horses. The 16S rRNA gene sequence (GenBank accession number AY527213) proved to be identical to the A phagocytophilum sequenced from Swedish horses as described by Johansson and others 28 and Engvall and others, 29 and to the HGE agent. The ankA and the groESL gene sequence (GenBank accession number AY529487 and AY529489, respectively) were compared to sequences deposited in GenBank.…”

GranulocyticEhrlichiainfection in Ixodid ticks and mammals in woodlands and uplands of the U.K.

“…PCR products from both habitats showed a high degree of homology with the agent of HGE, suggesting that human infections potentially could be acquired from tick bites in either of these U.K. habitats. Small differences between the 16S rRNA sequences of the samples from sites 1 and 2, and previously elucidated sequences of E. phagocytophila (Anderson et al, 1991), suggest some degree of genetic diversity of granulocytic Ehrlichiae in the U.K. as occurs in mainland Europe (Engvall et al, 1996). The differences between sequences from ticks of the upland (site 1) and sequences of E. phagocytophila isolated from sheep in Scotland (Anderson et al, 1991) suggest that U.K. sheep may themselves be competent reservoirs for a number of genotypes of granulocytic Ehrlichiae (Table 3).…”

“…Ͼ 99.7% homology of 16S rRNA sequences) suggest that E. phagocytophila, E. equi and E. microti and the HGE agent are conspecific (Anderson et al, 1991;Dumler et al, 1995;Telford et al, 1996;Sumner et al, 1997). Clearly, these organisms demonstrate some genetic diversity, at least in mainland Europe (Engvall et al, 1996), with potential consequences for variation in pathogenicity in different vertebrate species. However, the origins of such diversity is most likely to rest on ecological factors such as coevolution with natural reservoirs of both granulocytic Ehrlichiae and the vector tick.…”

Consensus Statement on Ehrlichial Disease of Small Animals from the Infectious Disease Study Group of the ACVIM*

“…PCR is a sensitive method for the detection of acute E canis and granulocytic ehrlichial infection in dogs. 51,52 PCR and DNA sequencing have been used to identify new species or to show that some Ehrlichia spp. such as HGE, E phagocytophila and E equi are closely related.…”